Anda di halaman 1dari 67

EBOOKS Cells in Tissues

FOR THE Christopher J. Paradise A. Malcolm Campbell


APPLIED BIOLOGY COLLECTION
Two systems illustrate how individual cells of an organ system
SCIENCES
function, communicate, and coordinate activities. The digestive
LIBRARY system breaks down and absorbs nutrients, and some special-
Create your own ized cells break down and absorb nutrients. The case of parietal
Customized Content cells in the stomach and epithelial cells in the small intestine

Cells in Tissues
Bundlethe more are used to describe how cells function as a unit within organ
books you buy, systems, coordinating activities and communicating with one
the greater your another. The endocrine system of insects affects molting and
discount! metamorphosis, and specialized cells are also important in each
of these processes within that organ system. The experiments
that were devised to determine the role of hormones in insect
THE CONTENT
molting and metamorphosis are described. Finally, stem cells
Energy Physics are healthy components of several different systems in animal
Engineering bodies and are described in relation to a disruption in function.
Biotechnology In this breakdown of function, cancer cells, in contrast to stem
Biology cells, can abnormally affect cell cycle regulation.
Mathematics
Christopher J. Paradiseis professor of biology and environ-
Chemistry
mental studies at Davidson College. He teaches introductory
biology, ecology, entomology, and topical seminars on ecotoxi-
THE TERMS cology and renewable natural resources. He also occasionally
Perpetual access leads a study abroad program in India. His research evaluates
for a one time fee anthropogenic factors that influence insect biodiversity at a
No subscriptions or variety of scales. His current research interests include effects of
access fees land use patterns on pollinator communities in parks.
Unlimited
A. Malcolm Campbellteaches biology at Davidson College,
concurrent usage
NC. He received national and international education awards:
Downloadable PDFs Genetics Society of America (2013); American Association for the
Free MARC records Advancement of Science (2012); and American Society for Cell
Biology (2006). He was the founding co-editor in chief of CBE Life
For further information,
Sciences Education; founding director of Genome Consortium
a free trial, or to order,
contact: for Active Teaching (GCAT); and member of the American Soci- Christopher J. Paradise
sales@momentumpress.net ety for Cell Biology governing council (20122014).
A. Malcolm Campbell
Cells in Tissues
Cells in Tissues

Christopher J. Paradise, PhD


A. Malcolm Campbell, PhD
Cells in Tissues
Copyright Christopher J. Paradise and A. Malcolm Campbell. 2016.

All rights reserved. No part of this publication may be reproduced, stored


in a retrieval system, or transmitted in any form or by any means
electronic, mechanical, photocopy, recording, or any otherexcept for
brief quotations, not to exceed 250 words, without the prior permission
of the publisher.

First published in 2016 by


Momentum Press, LLC
222 East 46th Street, New York, NY 10017
www.momentumpress.net

ISBN-13: 978-1-60650-951-7 (print)


ISBN-13: 978-1-60650-952-4 (e-book)

Momentum Press Biology Collection

Cover and interior design by S4Carlisle Publishing Services Private Ltd.,


Chennai, India

10 9 8 7 6 5 4 3 2 1

Printed in the United States of America


Abstract
Two systems illustrate how individual cells of an organ system function,
communicate, and coordinate activities. The digestive system breaks
down and absorbs nutrients, and some specialized cells break down and
absorb nutrients. The case of parietal cells in the stomach and epithelial
cells in the small intestine are used to describe how cells function as a
unit within organ systems, coordinating activities and communicating
with one another. The endocrine system of insects affects molting and
metamorphosis, and specialized cells are also important in each of these
processes within that organ system. The experiments that were devised to
determine the role of hormones in insect molting and metamorphosis are
described. Finally, stem cells are healthy components of several different
systems in animal bodies and are described in relation to a disruption in
function. In this breakdown of function, cancer cells, in contrast to stem
cells, can abnormally affect cell cycle regulation.

Keywords
multicellular organisms, undifferentiated cells, stem cells, tissues,
muscle cells, neurons, alimentary canal, information, ingestion, diges-
tion, assimilation, homestasis, stomach, small intestines, epithelial
cells, epithelium, smooth muscle, gastric pits, parietal cells, histamine,
proton pump inhibitor, tubulovesicles, pepsinogen, pepsin, peristalsis,
endocrine system, hormones, exoskeleton, molting, epidermis, hemo-
lymph, corpus allatum, metamorphosis, protocerebrum, prothoracic
gland, neurosecretory cells, stem cells, cancer cells, tumors, multipo-
tent, tumor suppressors, mitogen, central nervous system, peripheral
nervous system, carcinoma
Contents
Preface...................................................................................................ix
Acknowledgments....................................................................................xi
Introduction.........................................................................................xiii
Chapter 1 The Digestive System Breaks Down and Absorbs
Nutrients from Ingested Food............................................1
Chapter 2 Populations of Endocrine Cells in Animals Affect the
Whole Organism.............................................................17
Chapter 3 Similarities and Differences Between Stem Cells and
Cancer Cells.....................................................................27
Ethical, Legal, Social Implications: Decisions
Aboutto Whom and When Medical Interventions
are Given......................................................................38
Conclusion............................................................................................43
Glossary................................................................................................45
Index....................................................................................................49
Preface
This book about cells and how they are part of tissues and organisms
is part of a thirty book series that collectively surveys all of the major
themes in biology. Rather than just present information as a collection
of facts, the reader is treated more like a scientist, which means the
data behind the major themes are presented. Reading any of the thirty
books by Paradise and Campbell provides readers with biological con-
text and comprehensive perspective so that readers can learn important
information from a single book with the potential to see how the major
themes span all size scales: molecular, cellular, organismal, population
and ecologic systems. The major themes of biology encapsulate the
entire discipline: information, evolution, cells, homeostasis and emer-
gent properties.
In the twentieth century, biology was taught with a heavy emphasis
on long lists of terms and many specific details. All of these details were
presented in a way that obscured a more comprehensive understanding.
In this book, readers will learn about cells in tissues and some of the sup-
porting evidence behind our understanding. The historic and more recent
experiments and data will be explored. Instead of believing or simply
accepting information, readers of this book will learn about the science
behind cells in the context of tissues the way professional scientists do
with experimentation and data analysis. In short, data are put back into the
teaching of biological sciences.
Readers of this book who wish to see the textbook version of this
content can go to www.bio.davidson.edu/icb where they will find
pedagogically-designed and interactive Integrating Concepts in Biology
for introductory biology college courses or a high school AP Biology
course.
Acknowledgments
Publishing this book would not have been possible without the gener-
ous gift of Dr. David Botstein who shared some of his Breakthrough
Prize with co-author AMC. Davids gift allowed us to hire talented artists
(Tom Webster and his staff at Lineworks, Inc.) and copyeditor Laura
Loveall. Thanks go to Kristen Mandava of Mandava Editorial Services for
project management and guidance. In particular, we are indebted to Katie
Noble and Melissa Hayban for their many hours and attention to detail.
Kristen Eshleman, Paul Brantley, Bill Hatfield and Olivia Booker
helped us with technology at Davidson College. We are grateful to
administrators Tom Ross, Clark Ross, Carol Quillen, Wendy Raymond,
Verna Case, and Barbara Lom who had confidence in us and encouraged
us to persist despite setbacks along the way.
Thanks to my wife Amy Brooks for her constant support during the
development of this textbook, and my daughter Evelyn for her endless
energy. Thanks to Malcolm Campbell for his steadfast resolve and opti-
mism. Without him, this book would not exist. Thanks to collaborator
Laurie Heyer for taking my sometimes half-baked math ideas and turn-
ing them into powerful and elegant Bio-Math Explorations. I learned a
lot from both of them. While the math is largely absent from this book,
our collaboration with her made this a better book. Nancy Stamp at
Binghamton University, and Bill Dunson and Richard Cyr at The Penn-
sylvania State University influenced me greatly in how I think as a scientist
and approach my teaching. Finally, I thank my students in Integrated
Concepts in Biology II, who enthusiastically participated in our experi-
ment to redesign introductory biology, starting with the text and ending
with a new approach to teaching biology.
Introduction

Individual cells are affected by a variety of factors and can disrupt the
function of other cells. Despite the focus on the cell as an individual
entity, no cell exists in isolation and many biologists study cells in the
context of the whole organism. In this book, the focus is on the popula-
tion, but cells are viewed as populations within multicellular organisms.
Over evolutionary time, some cell populations of certain species evolved
into colonies of interdependent individuals. From those colonies, mul-
ticellular organisms evolved. Within a multicellular organism, popula-
tions of similar cells exist and function as a unit. They make up tissues
and organs. Each cell type exists as a population of cells, and each has a
particular structure related to function. Some cells remain undifferenti-
ated, waiting for the signal to become active and develop into a particular
type of cell. These stem cells have some properties that are similar to
cancer cells. The description of these phenomena reflect the themes of the
concept of cells: All cells come from preexisting cells, cells maintain inter-
nal environments that differ from external environments, cell structure
defines cell function, and cells communicate with other cells.
CHAPTER 1

The Digestive System


Breaks Down and
Absorbs Nutrients from
Ingested Food

In most multicellular organisms, cells make up tissues, which make up


organs, which make up organ systems. There may be various populations
of cells, or tissues, within one organ, and all the cells in the population
have similar structure and function. For instance, populations of muscle
cells respond similarly when neurons stimulate them. But the muscle
itself is made up of multiple types of cells. There are populations of con-
nective tissue cells, neurons, and other cells. Some of the cells within a
tissue are actually part of other systems. Neurons are part of the nervous
system and yet are found in all other systems in most animals. The cells,
tissues, organs, and systems are all highly interconnected, and the impact
of one population of cells upon an entire organism will be examined in
this book.
Even though cells at the tissue and organismal levels are investigated
in this book, whole organ systems will be considered to examine how
populations of cells may affect those systems. The digestive system is
an organ system that illustrates this. Specialized populations of cells
exist along the internal lining of the alimentary canal that are critical
in secreting digestive enzymes and absorbing nutrients. The alimentary
canal is a long tube in the body through which food passes after it is
ingested. Molecular information is detected by those cells, which trig-
gers release of enzymes and performs other functions as needed after a
meal is ingested. The processes of ingestion, digestion, and assimilation
2 CELLS IN TISSUES

are critical for maintaining homeostasis. Ingestion is the process of


taking food into the body. Digestion is the process of breaking down
food. Assimilation is the process of absorbing nutrients and incorporat-
ing them into the body. The components of the digestive system have
evolved in different animals for the particular diet of each animal. The
entire system is an emergent property that arises from the cell, tissue,
and organ components of the system.
The particular components of digestive systems of different animals
vary quite widely. Here, the human digestive system will be examined,
although we will examine data from closely related species in order to
understand the function of our own system. Early anatomists in the elev-
enth to fifteenth centuries (although Greek physicians began the practice
of dissection as early as 300 BCE), generated fairly accurate descriptions
and diagrams of the main structural components of the digestive system.
Often these anatomists worked on bodies of executed criminals, or they
worked in secret on cadavers due to the belief that souls of dissected bod-
ies could not go to heaven. Although many anatomists came before him,
Leonardo da Vinci provided detailed drawings of human digestive system
anatomy.
In large part, da Vinci did pretty well diagramming the positions of
the major organs of the human digestive system. The alimentary canal
and associated organs of the digestive system are all connected in many
ways to produce a functioning system that is an emergent property of its
component parts. The positions of the accessory glands and organs were
well-described by da Vinci.
Today the level of detail and knowledge of the anatomy, physiology,
and functions of the human digestive system is much greater than it was
500 years ago. The basic anatomy is well known (Figure 1). In general,
the mammalian digestive system consists of the alimentary canalalso
known as the gut, is the complete digestive tract through which ingested
food flows, is broken down, nutrients are assimilated, and waste is
expelled.
From mouth to anus, the alimentary canal of most mammals con-
sists of the esophagus, stomach, small intestine, and large intestine. In
humans, food (changing along the way) enters the mouth then passes to
the esophagus, stomach, small intestine, large intestine, and finally exits
THE DIGESTIVE SYSTEM BREAKS DOWN FOOD 3

salivary gland

pharanx tongue

salivary gland

esophagus

liver
stomach
gall bladder
pancreas
duodenum

large intestine jejunum

ileum

rectum
anus

Figure 1 Anatomy of the human digestive system.

the anus as solid waste, or feces. Accessory glands (also part of the diges-
tive system) secrete digestive juices into the canal through ducts. These
accessory glands consist of salivary glands, pancreas, liver, and its stor-
age organthe gallbladder. Data will be analyzed that were collected in
order to learn about the function of the specialized cells mentioned previ-
ously that aid in digestion and assimilation of nutrients.
Various organs within the digestive system function to assist with
digestion and assimilation. For instance, the esophagus is a tube that
primarily functions to transport food to the stomach, the stomach does
some digestion but is also a food storage organ, the small intestine is
where most assimilation occurs, and the large intestine is where the solid
waste is formed and stored until defecation. Some of these organs will
be examined at the cellular level to discover how populations of cells in
these organs function in the digestion and assimilation processes. The
digestive system of animals is quite complex with organs of the system all
interconnected and with the system connected to other systems, such as
4 CELLS IN TISSUES

the circulatory and nervous systems. In this chapter only a small portion
of the system will be examined in order to understand how cells function
within larger systems.
The process of digestion follows ingestion of food and begins with
the simple act of chewing. Food is then swallowed; it travels down the
esophagus and enters the stomach. For centuries, scientists struggled with
determining whether the stomach secreted an acid and if so what kind
of acid. In 1823, William Prout definitively answered this question for
several species of mammal. Prout quantified the acid by feeding rabbits,
horses, cows, and dogs and then immediately removing the contents of
their stomachs. (He had to kill the animals to do this.) He then repeatedly
exposed the contents to distilled water and combined the mixtures. Prout
divided the mixture into four equal portions. The first was evaporated
to dryness, burned at high temperature, and dissolved again in distilled
water. He used silver nitrate to react with the chloride salts in the solution
to determine the amount of chloride ion (Table 1). To the second portion
Prout used a known amount of potassium hydroxide to exactly neutralize
the solution, allowing him to determine the amount of free acid present.
In the third fraction, Prout added a large quantity of potassium hydroxide,
which will react and neutralize all hydrochloric acid (HCl) to potassium
chloride and water. He then determined total chloride in the same manner
as before, with silver nitrate. The final fraction was used to determine if
any other acids were found.
Prout did not find any other acid in stomach secretions. He was only
able to find HCl. Of course, as soon as it is known that the stomach pro-
duces HCl, scientists wondered where the HCl was made and how it was
produced given that HCl is so destructive and could potentially eat away
the stomach itself.

Table 1 Results from chloride analysis of stomach contents of three


rabbits.
fraction of solution rabbit 1 rabbit 2 rabbit 3
chloride salts (first fraction) (g) 0.12 0.95 1.71
exact amount of base to neutralize acid (second fraction) (g) 1.56 0.76 0.40
chloride salts after neutralization (third fraction) (g) 1.59 2.22 2.72
total amount of chloride (g) 3.27 3.93 4.83
other acids (fourth fraction) 0 0 0

Source: From Prout, 1823.


THE DIGESTIVE SYSTEM BREAKS DOWN FOOD 5

To address this question, something about the inner lining of the


stomach must be known. Careful microscopic examination led to knowl-
edge of the cellular makeup of the stomach wall. Like other organs, the
stomach consists of several types of cells. Most importantly, consider the
wall of the stomach to have several layers (Figure 2). The layer closest to
the lumen of the stomach, the inner open space, is called the mucosa.
Epithelial cells reside on the surface of the mucosa. Epithelial cells make
up the epithelium that encloses and protects organs and internal sur-
faces that have direct contact with the external environment. Inner to
the epithelium is connective tissue, which helps hold tissues and organs
together, and a thin layer of smooth muscle. The other layers, although
important to the functioning of the stomach, are not involved directly in
producing HCl. They consist of a submucosa with more connective tissue;
the muscularis externa beneath the submucosa, which consists of layers of
muscles arranged in different directions to help churn and move food;
and the serosa with more connective tissue.
Scientists discovered several cell types along the epithelium and that
the entire epithelium is arranged in convoluted pits called gastric pits.

stomach surface epithelium

gastric pit

mucosa
gastric gland

submucosa parietal
cell
muscularis
externa chief cell

serosa

enteroendocrine cell

Figure 2 Layers and cell types of the stomach. In the epithelium,


gastric pits lead to gastric glands that secrete gastric juice. The
gastric glands (one gland is shown enlarged on the right) contain
different types of cells that secrete a variety of enzymes, including
HCl, which activates the protein-digesting enzyme, pepsin.
6 CELLS IN TISSUES

These gastric pits are indentations that lead to the gastric glands from
which the secretions come. The human stomach has several million of
these pits, and further into each pit scientists discovered other types of
cells, which have different functions; one of which will be examined.
Figure 2 shows parietal cells, located in pits in the upper part of the
stomach, which are the cells that produce HCl. Any macromolecule that
is affected by acidic conditions can begin to break down in the stomach
after HCl is secreted. The parietal cells thus have an important role in the
digestion of food.
When a person eats, their stomach becomes distended and proteins
begin to be broken down into component amino acids; substances are
released that activate parietal cells (Figure 3). These substances include
gastrin, histamine, and acetylcholine. Activation leads to a series of events
at the molecular level within parietal cells.
Muallem and colleagues performed a study to determine how stim-
ulation of the parietal cells affected parietal cell internal pH. If pH of
individual cells goes up, then it can be concluded that hydrogen ions are
being excreted from the cell and pH of the surrounding environment goes
down. The scientists used a centrifugation technique to isolate parietal
cells from the epithelium of the upper part of a rabbits stomach. Keep
in mind the structure of the parietal cell, especially the deep infolding on
the lumen end of the cell, which increases the surface area for secretion.
The measurement of internal cell pH in parietal cell suspensions was
achieved through use of a dye that can permeate cells. This dye can be used

neuron

A parietal cell

A
G cell
ECL
neuron
cell HCl
H

G G
circ
ula
tion

Figure 3 Activation of parietal cells.


THE DIGESTIVE SYSTEM BREAKS DOWN FOOD 7

to estimate pH inside the cells. The scientists measured the emission inten-
sity of the dye at two different wavelengths; the ratio of the intensities is
pH-dependent. Knowledge of the intensity ratio in solutions of known pH
allows researchers to estimate intracellular pH. The scientists incubated cells
with dye for 20 minutes and then washed the cells so that any dye not taken
up by cells was washed away. Keep in mind that relatively small changes in
pH, which is measured on a logarithmic scale, in single cell suspensions
could actually lead to large decreases in pH in the stomach lumen.
Muallem and colleagues exposed cells to a medium that contained no
sodium ions (Na+) and found that intracellular pH went down (Table 2).
When they added Na+, the pH rapidly went up. Addition of a chemical
that inhibited the Na+/H+ exchange protein at the same time that Na+
was added completely inhibited the rise in pH, illustrating that Na+/H+
exchange is important for maintaining intracellular pH in the resting cell.
Next, Muallem and colleagues exposed cells to histamine, a known
activator of parietal cells (Table 2). The Na+/H+ exchange inhibitor was
added in one treatment before exposure to histamine. Histamine binds

Table 2 Intracellular pH changes from experiments on


parietal cells. Negative values indicate declines in pH.
treatment pH
no Na+ 0.58
Na+ added after exposed to no Na+ 0.56
+ + +
Na and Na /H exchange inhibitor added after
0
exposed to no Na+
histamine 0.130 0.038
no Na+, then histamine 0.04
+ +
Na /H exchange inhibitor added, then histamine 0.04
histamine receptor antagonist, then histamine 0.01
histamine receptor antagonist, then histamine,
0.125 0.027
then chemical that increases cAMP
histamine receptor antagonist, then histamine,
then Na+/H+ exchange inhibitor, then chemical 0.01
that increases cAMP
H+/K+ATPase inhibitor, then histamine 0.09
no Na+, H+/K+ATPase inhibitor, then histamine 0
Na+/H+ exchange inhibitor added, then
0.02
H+/K+ATPase inhibitor, then histamine
Source: From Muallem et al., 1988, text and Figures 2 to 8.
8 CELLS IN TISSUES

to a receptor on parietal cells. A chemical known to block histamine-


receptor was added (an antagonist), after which histamine was added.
Binding of a ligand (such as, histamine) to a receptor often activates the
second messenger cyclic adenosine monophosphate (cAMP). A chemical
that raises cAMP levels in cells was added after action of histamine was
blocked by its antagonist to determine if histamine increased cAMP in
parietal cells (Table 2). If the Na+/H+ exchange inhibitor was also added,
there was essentially no change in intracellular pH.
Finally, an H+/K+-ATPase inhibitor was found to not substantially
affect histamine activation, but if there was no Na+ in the cell medium,
there was no effect of histamine. If the cells were exposed to the Na+/
H+ exchange inhibitor, there was no activation (Table 2). The H+/K+-
ATPase inhibitor is also called a proton pump inhibitor (PPI), and these
drugs are used to treat acid-related diseases, such as peptic ulcer disease.
In the absence of extracellular Na+ or in the presence of the Na+/H+
exchange inhibitor, intracellular pH increased only a very small amount.
The major contribution to the rise in intracellular pH is activity of the
Na+/H+ exchange protein. Prior to stimulation by histamine, gastrin,
or acetylcholine, parietal cells use Na+/H+ exchange to maintain pH
homeostasis. Most eukaryotic cells do this. Na+/H+ exchange proteins
are located on the side of the cell away from the lumen, and this should
make sense for a protein that is simply maintaining pH and not excret-
ing it into the stomach lumen. That end of the parietal cell also contains
Cl/HCO3 exchange proteins. These will become important when the
parietal cell is stimulated.
Upon stimulation, parietal cells secrete more acid than any other type
of cell in the body. Each activating chemical, histamine, gastrin, and ace-
tylcholine has a receptor on the parietal cell. Gastrin, which is released
from G cells in response to stretching of the stomach, nervous system
impulses, and presence of amino acids, also has a receptor on the special-
ized cells that secrete histamine and is what causes release of histamine.
The data suggest that histamine by itself produces a large increase in intra-
cellular pH and that it is in fact histamine having the effect, because
presence of the histamine receptor antagonist negates the effect of hista-
mine. Histamine has the effect of increasing cAMP levels in the parietal
cells, and this is because the effect of histamine receptor antagonist, then
THE DIGESTIVE SYSTEM BREAKS DOWN FOOD 9

histamine, and then the chemical that increases cAMP leads to a large
increase in intracellular pH. This suggests that the histamine-histamine
receptor interaction leads to an increase in cAMP levels in the cell.
The increase in cAMP levels within the parietal cell leads to other
changes. The entire scheme is complex and not of it all is shown here, but
ultimately acid secretion at the lumen end of the parietal cell occurs. The
Cl/HCO3 exchange of proteins at the end of the cell away from the
lumen is responsible for expelling HCO3 and bringing in Cl.
Recall that in the absence of Na+, intracellular pH dropped dramati-
cally because the parietal cell could not rid itself of excess H+. In the
presence of Na+, the resting, non-stimulated cell has an intracellular
pH of about 7.1. In the absence of Na+ but the presence of the H+/
K+-ATPase inhibitor and histamine, there is a slight rise in intracellular
pH. This rise is larger when there is just Na+ and histamine but only
after a lag period. The researchers concluded that the H+/K+-ATPase
was responsible for the further rise in pH to about 7.3. The action of
this pump expelling H+ into the lumen is what leads to production
of HCO3. Expelling HCO3 brings Cl into the parietal cell, which
then diffuses to the lumen end and is then secreted into the lumen. The
K+ also leaks back out through a separate channel protein after being
exchanged with H+.
The acid secretion at the lumen end of the parietal cell results
in higher activity of anion exchange, which is caused ultimately by
the higher intracellular pH due to higher HCO3. This leads to main-
tenance of cell homeostasis; researchers have found that after the initial
increase in intracellular pH, if the parietal cell continues to secrete H+
and Cl, intracellular pH does not continue to rise. The combined
function of both exchangers is necessary for optimal acid secretion.
The parietal cell clearly has specialized proteins to facilitate the pro-
duction of acid. It also has a specialized morphology (Figure 4). The rest-
ing state looks very different from the stimulated state.
In the resting state, deep infoldings on the lumen end of the parietal
cell are seen. The H+/K+-ATPase pumps are sequestered within vesicles
called tubulovesicles. Activation of acid secretion leads to two func-
tional changes: tubulovesicles fuse with the membrane of the infolded
region that opens to the stomach lumen, and this membrane acquires
10 CELLS IN TISSUES

tubulovesicles

at rest

mitochondria

infolding

stimulated
nucleus
infolding

Figure 4 Schematic representation of parietal


cells at rest and under stimulation.

permeability to KCl, which allows both K+ and Cl to enter the lumen,


as shown earlier.
The stomach produces other substances, including the protein pepsino-
gen, which breaks down to pepsin in the presence of acid. Pepsin cleaves
proteins in ingested food. We have known about pepsin since 1836 when The-
odor Schwann described a water-soluble factor in gastric juice that digested
egg white. Mucus is also secreted by other cells. HCl, pepsinogen, and mucus
together make up gastric juice. The stomach lining is protected from gas-
tric juice by the epithelial cells, which produce and secrete a bicarbonate-rich
solution and form a HCO3-rich mucus layer that coats the mucosa and
protects the epithelial cells. Bicarbonate is alkaline (a base), and it neutralizes
the acid secreted by the parietal cells, producing water in the process. This
continuous supply of bicarbonate is the main way that the stomach protects
itself from digesting itself and the overall acidic environment.
Peristalsis, a rhythmic contraction of muscles of the alimentary tract,
moves the partially digested food from the stomach into the small intestine.
The small intestine is made up of three partsthe duodenum, jejunum,
THE DIGESTIVE SYSTEM BREAKS DOWN FOOD 11

absorptive cells capillary


artery
vessel vein
carrying
blood
villi

lumen

muscle
layers lymphatic
villi vessel

lumen of small intestine

Na+ glucose 2K+ H2O


osmosis
Na+/K+-
ATPase

GLUT-2
transport
protein

glucose blood 3Na+ H2O osmosis

Figure 5 Small intestine epithelial cells and glucose transport.

and ileum, in that order. Digestion is completed, and assimilation of the


components of food (sugars, amino acids, and fats) occurs in the small
intestine. As with the stomach, the small intestine has populations of spe-
cialized cells that absorb nutrients. Most enzymes that break down macro-
molecules in the lumen of the small intestine are secreted by the pancreas.
As with the stomach and most other organs, the small intestine is
lined with epithelial cells of various types that play specialized roles in the
digestive process (Figure 5). Many of these cells play multiple roles and
can take up a variety of nutrients. The cells involved in glucose transport
will now be examined.
The three major classes of nutrientsproteins, lipids (fats), and
carbohydratesall undergo digestion in the lumen of the small intes-
tine. Proteins are degraded into peptides and amino acids before absorp-
tion. Lipids (fats) are degraded into fatty acids and glycerol. Some
12 CELLS IN TISSUES

carbohydrates are degraded into simple sugars or monosaccharides, such


as glucose. Enzymes break down starch to shorter oligosaccharides, and
then enzymes located on the lumen end of epithelial cells break these
short chains into monosaccharides.
The inner wall of the small intestine is also called the mucosa and is
lined with epithelial cells. Structurally, the mucosa is highly wrinkled.
Microscopic villi project from these folds, and capillaries project into the
villi from outside the small intestine. Capillaries are small blood vessels
that form a network between arterioles and venules in the circulatory sys-
tem. Nutrients, vitamins, and even toxins, pass from the lumen through
epithelial cells, into the inside of the villi, and then into the capillaries
or lymphatic vessels where they are transported to the rest of the body.
Lymphatic vessels are thin walled, valved structures that carry lymph,
a colorless fluid that contains white blood cells. This movement occurs
through diffusion or active transport. Individual epithelial cells also have
finger-like projections known as microvilli. These adaptations increase the
surface area of the small intestine. Undigested and unabsorbed material
passes into the large intestine.
Pia Rder and her colleagues studied glucose absorption and trans-
port in small intestine epithelial cells in mice. They took advantage of
mutant strains of mice that lacked the ability to produce two trans-
membrane proteins known to transport glucose: the sodium-dependent
glucose cotransporter (SGLT1) and glucose transporter 2 (GLUT2). The
researchers bred sglt1 wild-type (sglt1+/+), sglt1 mutant (sglt1/), glut2
wild-type (glut2+/+), and glut2 mutant (glut2/) mice.
Mice were fed a solution with radioactive 14C glucose. The research-
ers standardized the amount given to mice based on body mass. After
15 minutes the animals were euthanized, blood was collected, and
the small intestine was dissected out, everted, and washed thoroughly
for analysis. Blood was centrifuged and analyzed for radioactive 14C
(Figure 6). Sections of small intestine were used to measure incorpo-
rated radioactivity, which was used to determine glucose retention
(see Figure 6).
Sections of the epithelium of the jejunum were incubated with anti-
bodies for SGLT1 or GLUT2 and stained for cell nuclei. This allowed
the researchers to visualize localization of SGLT1 and GLUT2 along the
THE DIGESTIVE SYSTEM BREAKS DOWN FOOD 13

tracer in plasma (dpm/10 ml)


350 ** 900 **

over 15 min. (nmol/cm)


glucose accumulation
300 750
250
600
200
450
150
300
100
50 150

0 0
A sglt1+/+ sglt1/ B sglt1+/+ sglt1/

tracer in plasma (dpm/10 ml)


750 * 1400 **
over 15 min. (nmol/cm)
glucose accumulation

1200
600
1000
450 800

300 600
400
150
200
0 0
C glut2+/+ glut2/ D glut2+/+ glut2/

Figure 6 Effects of SGLT1 and GLUT2 on glucose accumulation


and blood plasma glucose, as measured by amount of radioactive
tracer. Bars on left are wild-type and bars on right are mutant mice.
Error bars represent 1 standard error (SE). Statistical analyses
were performed using a t-test. *p-value 0.05. **p-value 0.01.
n = 4 to 6 mice per group. A, Mean accumulation of glucose in
intestinal tissue samples for sglt1 wild-type and mutant mice. B,Mean
amount of glucose in blood plasma for sglt1 wild-type and mutant
mice. C, Mean accumulation of glucose in intestinal tissue samples
for glut2 wild-type and mutant mice. D, Mean amount of glucose in
blood plasma for glut2 wild-type and mutant mice.
Source: Rder et al. 2014, Figures 1 and 3. PLoS One 9(2): e89977. doi:10.1371/journal.pone.
0089977. 2014 Rder et al.

epithelial cell membranes (Figure 7). In the images, rows of epithelial cells
that line up to form the villi of the small intestine can be seen (see also
Figure 5). Note where SGLT1 localizes relative to the cell nuclei and how
that differs from the localization of GLUT2.
By knocking out the sglt1 gene in mice and then comparing
responses to wild-type mice, scientists can determine the effect of the
SGLT1 transmembrane protein. This is also true for the glut2 mutants.
14 CELLS IN TISSUES

A B

C D

Figure 7 Immunostained cells from wild-type and mutant mice


jejunum epithelial cells showing localization of transmembrane glucose
transport proteins (gray shading) and cell nuclei (white/light gray
shading; used for reference). A, Localization of SGLT1 from sglt1+/+
wild-type mice. Two villi are shown with rows of epithelial cells.
Bright blue nuclei are cells within the villi, not the lumen. Note
that SGLT1 localizes away from the nucleus along the membrane
in contact with the lumen. B, Localization of SGLT1 from sglt1/
mutant mice. One villi is shown. C, Localization of GLUT2
from wild-type glut2+/+ mice. A portion of one villi is shown. D,
Localization of GLUT2 from mutant glut2/ mice. Portions of three
villi are shown.
Source: Rder et al., 2014, Figures 5 and 6. PLoS One 9(2): e89977. doi:10.1371/journal.pone.
0089977. 2014 Rder et al.

Mutantsglt1/ mice take up very little glucose into epithelial cells and
show very little glucose in the blood relative to wild-type mice. The scien-
tists concluded that the function of SGLT1 was to transport glucose into
the epithelial cell. If glucose is not getting into the epithelial cell, then it
cannot find its way into the blood. The localization of SGLT1 near the
lumen further confirms this. The lack of SGLT1 in the mutants confirms
that the mutant really is deficient in producing that protein.
THE DIGESTIVE SYSTEM BREAKS DOWN FOOD 15

Glucose accumulation in epithelial cells is higher in mutant glut2/


mice than wild-type mice. But glucose concentrations are lower in the
blood of those mutant mice than in the wild-type mice. This suggests
that glucose can enter the epithelial cell but cannot get out. So it enters
but doesnt leave the epithelial cells in mutant glut2/ mice. It enters and
leaves normally in wild-type mice, and the response is similar to the
sglt1+/+ mice. GLUT2 localizes in membranes on the sides and away
from the lumen, appearing to surround the nucleus of epithelial cells (see
Figure 7). Again, the mutant is deficient in GLUT2.
In summary, epithelial cells of the small intestine possess two trans-
membrane proteins: one that is sodium-dependent and brings glucose
into the cell from the lumen, and one that transports it into the villi to
be picked up by the blood capillaries. These epithelial cells possess many
other specialized proteins that are used to take up other sugars, amino
acids, fatty acids, and other nutrients. This is convincing evidence for the
existence and function of two of these transmembrane proteins.
To complete this short tour of the digestive system, material that has
not been digested and absorbed by the small intestine enters the large
intestine. Here fermentation of any remaining digestible matter by the
gut flora occurs. Any remaining semisolid waste (termed feces) is removed
by the coordinated contractions of the intestinal walls (peristalsis), which
propels it forward to reach the rectum and exit via defecation from the
anus. Specialized cells occur in this organ too, although they will not be
examined here. Data on only a small part of the mammalian digestive sys-
tem has been explored here. Specialized populations of cells exist in each
organ and function to break down and absorb various nutrients into the
body. In the next chapter, populations of cells of another organ system,
the endocrine system, and how they also affect the whole organism, will
be examined.

Bibliography
Baron JH: The discovery of gastric acid, Gastroenterology 76:10561064,
1979.
Baumont W: Experiments and observations on the gastric juice and physiol-
ogy of digestion, Cambridge, MA, 1929, Harvard University Press.
16 CELLS IN TISSUES

Rder PV, Geillinger KE, Zietek TS, et al.: The role of SGLT1 and GLUT2
in intestinal glucose transport and sensing, PLoS One 9(2):e89977,
2014.
Kousoulis AA, Tsoucalas G, Armenis I, et al.: From the hungry acid to
pepsinogen: a journey through time in quest for the stomachs secre-
tion, Ann Gastroenterol 25(2):119122, 2012.
Langley JN, Edkins JS: Pepsinogen and pepsin, J Physiol 7(5-6):371415,
1886.
Prout W: On the nature of acid saline matters usually existing in the
stomachs of animals, Philos Trans R Soc Lond 1:4549, 1824.
CHAPTER 2

Populations of Endocrine
Cells in Animals Affect the
Whole Organism

Recall that in most multicellular organisms, cells make up tissues, which


make up organs, which make up organ systems. There may be various
populations of cells, or tissues, within one organ as just learned in the
study of the digestive system. The cells, tissues, organs, and systems are
highly interconnected. One organ system is highly connected to other
systems, and often closely associated with the nervous system is the
endocrine system. Different groups of animals have different configura-
tions of endocrine systems; many have specialized populations of cells
within other systems that secrete hormones, an important component
of endocrine systems. Hormones are chemical substances, usually pep-
tides or steroids, produced by one cell type, that control and regulate
activity of other cells. Advances in our knowledge of how hormones
affect growth and development of animals have come from biologists
who have performed clever manipulations to determine the role of vari-
ous cell populations in the regulation and secretion of hormones.
Vincent Wigglesworth performed some of the crucial work piecing
together key aspects of the growth of insects. Insects are invertebrate ani-
mals that have a jointed exoskeleton, one pair of antennae, and six legs.
An exoskeleton is a hardened external supportive covering composed of
nitrogen-containing polysaccharides, protein, and lipids. How do animals
grow bigger if they have an exoskeleton? They must either add to it so that
it expands, or grow a new, bigger one and shed the old one. Insects do the
latter. What regulates the molting, or shedding of the old exoskeleton, of
insects is one of the questions that intrigued Wigglesworth.
18 CELLS IN TISSUES

One of the model insects he used was Rhodnius prolixus, which is a


blood-sucking bug found in South America. R. prolixus grows into the
adult by molting five times, each molt being preceded several weeks ear-
lier by a meal of blood. One of the advantages of studying this insect is
that it only requires one blood meal in between each molt.
Wigglesworth took advantage of the fact that R. prolixus takes only
one blood meal in between each molt and performed a simple experi-
ment. On each day during the period of time after feeding, the biologist
decapitated a number of insects. He did this for insects in each of the five
juvenile stages. After decapitation, the wound was sealed with paraffin wax
to prevent fluid loss. He then observed the effect on molting (Table 3).
The graphs show the percentage of individuals decapitated on the indi-
cated day after taking a blood meal that eventually molted, not the day on
which they molted. The molt occurred days after the decapitation, if at all.
Wigglesworth then took a group of individuals in the fourth stage,
fed them all on the same day, and decapitated half of them 24 hours
later. The other half served as controls. On each succeeding day, he took
one decapitated and one control individual and removed part of the
exoskeleton and underlying epidermis from each. An epidermis is an
outer layer of cells in multicellular organisms. He stained these tissues in
order to compare the cellular state of the epidermis. The scientist found
that epidermal cell cytoplasm in normal individuals became denser and
deeply staining over time, and then after 5 days the cells began to divide.
In decapitated insects, these changes were absent. Other cells associated

Table 3 The proportion of R. prolixus that molted in each stage after


being decapitated on different days post-feeding. Molting occurred days
after the decapitation.
number of days after percent of percent of percent of percent of percent of
feeding at which 1st stage 2nd stage 3rd stage 4th stage 5th stage
decapitation occurred molting molting molting molting molting
2 0 0 0 0 0
3 50 60 83 50 0
4 100 80 87 60 0
5 100 83 100 0
6 100 100 85 20
7 100 100 50
8 100 85
9 100

Source: From Wigglesworth, 1934, Figure 1.


POPULATIONS OF ENDOCRINE CELLS AFFECT ANIMALS 19

with the epidermis did not differ developmentally immediately after


decapitation, compared to control insects.
Next, Wigglesworth allowed six fifth stage individuals to blood-feed
and decapitated them 7 days later. The biologist examined the epidermis
of these individuals and found that they were growing new epidermal
cells through mitosis in preparation for molting.
In each stage, there is what Wigglesworth called a critical perioda
period of time during which the head is necessary for molting. After that
critical period is over, the head is no longer necessary for molting. Because
the length of time of each stage varies, the critical period also varies. The
first stage is shortest, and it has a shorter critical period than other stages.
Furthermore, the biologist was able to show that the epidermis begins
to change shortly after blood feeding. Epidermal changes are necessary
for molting; the epidermis secretes the new exoskeleton that replaces the
old one. Mitosis in the epidermis begins several days after blood feeding,
and other epidermal changes begin to occur. Insects decapitated 1 day
after blood feeding did not exhibit those changes. Epidermal changes and
molting occurred in fifth stage individuals that were decapitated after 7
days. Wigglesworth concluded that the head is necessary for a growth or
molting hormone and that this hormone increases in concentration over
time. After it reaches a critical concentration that initiates mitosis in the
epidermis, the head is no longer necessary for molting to occur.
Wigglesworth reasoned that if this conclusion was correct, he ought
to be able to induce molting in an insect that had not passed the critical
period by exposing it to hemolymph, the body fluid of insects, of an
insect that had passed the critical period. In order to test this, the biolo-
gist decapitated insects and connected them (Figure 8). For each con-
nected pair, one individual had passed through the critical period and
one had blood fed only 24 hours earlier. He fastened them together with
paraffin wax so that the hemolymph could flow freely from one insect
to the other. He performed this experiment many times using insects in
every stage. Data for post-critical period fourth stage individuals cross-
circulated with fourth or fifth stages decapitated precritical period (24-
hour) is shown in Table 4.
There is much variation inherent in biological systems, and some of
that variation is seen in Table 4. Fourth stage individuals molt if they
20 CELLS IN TISSUES

fourth instar R. proxilus fourth instar R. proxilus


connected to fifth instar connected to fourth instar
Figure 8 Decapitated R. prolixus bugs
attached to each other.
Source: From Wigglesworth, 1934, Figures 2 and 3, Plate 14.

Table 4 Molting of fourth or fifth stage R. prolixus individuals


that were decapitated 24 hours after feeding and then connected
to another decapitated fourth stage individual that had passed the
critical period.
number of days after feeding that percent of
4th stage insects were connected all insects
to precritical period stage insects molting
5 54.5
6 81.0
7 90
8 100
9 100
10 100
12 100
Source: From Wigglesworth, 1934.

are decapitated on the fifth day after blood feeding, according to Table 3,
but they do not all have the ability to induce molting in fourth stage
individuals decapitated after 24 hours. Most 24-hour decapitated indi-
viduals molted with the individual to which they were connected if that
individual was decapitated at or after the critical period. Wigglesworth
concluded that either there is a molting hormone secreted from the head
or nervous impulses that cause secretion by a gland posterior to the head.
Wigglesworth knew of several glands that were candidates for the
source of hormone. He took serial sections of the head and thorax of
fifth stage insects in all phases of the molting processfrom fasting to
10 days after feeding, and then from every other day until molting. Serial
POPULATIONS OF ENDOCRINE CELLS AFFECT ANIMALS 21

sections are thin cross sections of an organism, in a series, from front to


back or top to bottom. The insect tissues were embedded in wax, which
allowed very thin sections to be sliced. The sections were then stained
so that structures containing nucleic acids (such as, ribosomes and the
cell nucleus) were stained a blue-purple hue and structures composed of
mostly protein were stained bright pink. Wigglesworth examined the sec-
tions from insects sectioned on different days post-feeding and found that
there was only one organ that showed a definite cycle of change in cells
that coincided with the critical period, the corpus allatum (plural: cor-
pora allata). The cytoplasm in the cells of the section made 7 days post-
feeding mostly stained bright pink, and although the cytoplasm stained
pink in a section taken 12 days post-feeding, the extent of staining was
different. Wigglesworth noted that these sections were similar to sections
taken before day 5.
Although some other gland that did not change at the cellular level
could have been the source of the hormone, cellular changes are often
associated with the production and secretion of proteins and hormones.
Therefore, Wigglesworths observations suggest that the source of the hor-
mone was the corpus allatum.
In the same research paper, Wigglesworth described other experi-
ments to determine how hormones affected metamorphosis. Metamor-
phosis is an abrupt developmental change in the form or structure of an
animal. Insects go through several juvenile stages, as discussed earlier,
which are separated by molts. The final molt results in the adult insect.
Wigglesworth was interested not only in the hormone that controlled
molting but also in how the endocrine or nervous system controlled
metamorphosis, and he was also interested in how the insect knew
whether to molt into a bigger juvenile or an adult, complete with wings
and mature sex organs.
The biologist performed more experiments on R. prolixus, this time
focusing on the conditions that cause molting into the adult stage. To
test whether there is a hormone in the hemolymph that causes meta-
morphosis, Wigglesworth again connected two decapitated individuals.
One individual in the pair was a fifth stage individual that had passed
the critical period, and the other was a fourth stage insect that had been
decapitated 24 hours after feeding prior to the end of the critical period.
22 CELLS IN TISSUES

He found that both insects in the pair molted simultaneously and that
fourth stages molted into the fifth stage but with adult characteristics. He
then repeated the experiment using fifth stage individuals that had passed
the critical period connected to first stage individuals decapitated 24
hours after feeding. When these first stages molted into the second stage,
Wigglesworth found two-thirds of them to be diminutive adults.
In the preceding experiments (see Table 4) where fourth stage indi-
viduals decapitated after the critical period were connected to fifth stage
individuals decapitated 24 hours after feeding, the fifth stages that molted
became adults, but there were defects in the adult structures, most nota-
bly in genitalia that were imperfectly formed. The further past the critical
period the fourth stage individuals were, the stronger the effects were on
the development of adult structures as the fifth stage individuals molted
into adults. In a final set of experiments, fourth stage individuals that had
just reached the critical period were connected to each other, and it was
found that both individuals developed some adult characteristics when
they molted into the fifth stage.
When earlier stages past the critical period were connected to fifth
stage individuals decapitated 24 hours after feeding, the fifth stage indi-
viduals almost always molted, as seen in Table 4. This suggests that the
molting hormone is the same across all stages of development. Thus
there is a second hormone whose presence prevents metamorphosis in
early stages or whose absence causes metamorphosis in the fifth stage.
In fact, the hormone present in fourth stages past the critical period
caused defects in fifth stage individuals molting into adults, indicating
that it is the presence of the hormone, not its absence that prevents
molting from juvenile to adult. It did not prevent those fifth stage indi-
viduals from molting into adults, however, due to the difference in size
between fourth and fifth stages. Because fifth stage individuals are two
to three times larger than fourth stages, the cross-circulated blood is
diluted in the fifth stage. Thus, it is the absence of the hormone that
causes metamorphosis.
Wigglesworths next task was to determine the source of the hor-
mone, which is now called juvenile hormone. He performed decapitation/
connection experiments (Table 5) and examined serial sections of early stage
individuals as he did with fifth stage individuals. Recall that in fifth stage
POPULATIONS OF ENDOCRINE CELLS AFFECT ANIMALS 23

Table 5 Decapitation experiments of R. prolixus individuals


to determine the source and action of juvenile hormone.
c.p. = critical period.
first R. prolixus second R. prolixus results
fourth stage decapitated 24 hours fourth stage decapitated 24
no molting
after feeding hours after feeding
fourth stage post-c.p., with only fourth stage decapitated 24 all pairs molted normally to
tip of head cut off hours after feeding fifth stage
all pairs molted normally to
fourth stage decapitated post-c.p. fourth stage decapitated post-c.p.
fifth stage
most metamorphose to fifth
fourth stage decapitated right at c.p. fourth stage decapitated right at c.p.
stage adult
fourth stage post-c.p., with only tip of fifth stage decapitated 24 hours fifth stages molted into an
head cut off after feeding abnormal sixth stage
fourth stage post-c.p., with tip of head
fifth stage decapitated 24 hours fifth stages molted into an
and all of brain removed, but corpus
after feeding abnormal sixth stage
allatum remained
fifth stage post-c.p. decapitated fourth stage post-c.p. with brain only all individuals molted into
behind corpus allatum removed another juvenile stage

Source: From Wigglesworth, 1934.

individuals during the critical period, cells in the corpus allatum become
swollen and stained pink. The scientist found that in earlier stages, a smaller
proportion of cells in the corpus allatum exhibited the change observed in
fifth stages. The remaining cells do not undergo this particular change.
The experiments described in Table 5 suggest that the head, specifi-
cally the corpus allatum, is the source of the hormone that regulates meta-
morphosis and that the hormone is different from molting hormone. If
just the tip of the head is cut off in a post-critical period individual, juve-
nile hormone is still produced, as indicated by the effects on individuals
decapitated prior to reaching the critical period. It appears that the head
is still necessary after the critical period in order to prevent metamorpho-
sis, and this is further supported by decapitation experiments on fourth
stages at compared to beyond the critical period.
The serial section results that differed between early stages and fifth
stages led Wigglesworth to conclude that the corpus allatum was not
only the source of molting hormone but was also the source of juvenile
hormone. Earlier it was stated that all cells swell and stain pink in the
fifth stage corpora allata. This is likely due to the high concentration of
molting hormone necessary to induce molting in a larger individual.
Because all cells swell, the scientist reasoned that all were producing
molting hormone; none were producing juvenile hormone. Wiggles-
worth further found that cells from the corpus allatum in early stage
24 CELLS IN TISSUES

individuals did not all stain pink, and he suggested that cells that do
not stain pink in early stages are the source of juvenile hormone. Later
experiments where he transplanted the corpus allatum from an individ-
ual past the critical period to a fifth stage that was decapitated 24 hours
after feeding confirmed this: Those fifth stages molted into another
juvenile stage, not the adult.
Although Wigglesworth hypothesized that the source of molting
hormone was the corpus allatum, he still need to perform experiments
to support his hypothesis. Several years after his initial experiments on
molting and metamorphosis, which were conducted in the early 1930s,
he performed more experiments on molting hormone. In these experi-
ments, Wigglesworth used more sophisticated dissection techniques than
the decapitation experiments conducted earlier. In these experiments,
he dissected out different parts of the brain, including different lobes,
nerves, and ganglia, masses of nerve tissue containing cell bodies of neu-
rons, from fourth or fifth stage individuals past the critical period and
transplanted those organs into the abdomens of fourth stage individuals
that were decapitated 24 hours after feeding (thus, they had not passed
the critical period; Table 6).
As stated earlier, individuals decapitated early do not molt unless they
are artificially provided with molting hormone. Wigglesworth reasoned

Table 6 Brain transplant experiments of R. prolixus individuals


to determine the source of molting hormone. In each experiment,
the indicated organ or part of the brain was transplanted from a
post-critical period R. prolixus and transplanted to a fourth stage
precritical period R. prolixus.
4th stage precritical
organ or part of brain results
period transplantee
whole brain decapitated 84% molted
dorsal half of central brain (above esophagus) decapitated 100% molted
small dorsal fragment of the protocerebrum decapitated 66.7% molted
corpus allatum and nearby brain ganglion decapitated none molted
ventral half of central brain (below esophagus) decapitated none molted
optic lobe decapitated none molted
subesophageal ganglion decapitated none molted
entire brain below protocerebrum decapitated none molted
head and first thoracic
small dorsal fragment of protocerebrum none molted
segment removed

Source: From Wigglesworth, 1940.


POPULATIONS OF ENDOCRINE CELLS AFFECT ANIMALS 25

that if molting could be induced in any of these treatments, it would


provide evidence for the source of molting hormone.
This set of experiments conclusively demonstrates that the corpus
allatum is not the source of molting hormone. Rather, it appears that
there is a small portion of the protocerebrum, located in the dorsal half
of the brain that controls molting. There is not likely to be nervous system
control by these cells because they were transplanted into the abdomen
of other insects, so it is a chemical, not a nervous signal, which caused
the molting. It is known that there are neurosecretory cells in parts of
the insect brain, and in other animals as well, that have both nervous and
endocrine functions. Neurosecretory cells are specialized neurons that
respond to stimulation by producing and secreting specific chemical mes-
sengers. The protocerebrum contains such cells. Wigglesworth concluded
that the neurosecretory cells in the protocerebrum mediated the secretion
of molting hormone from a gland in the thorax.
The gland was later discovered and named the prothoracic gland,
which was already known in other species of insect. Because of the diffuse
nature of this gland, Wigglesworth overlooked it in his earlier studies,
erroneously concluding that the corpus allatum was responsible for molt-
ing hormone. Experiments performed since Wigglesworths groundbreak-
ing studies confirmed his observations and revealed that neurosecretory
cells are stimulated after a blood meal and do secrete a hormone that
stimulates the prothoracic gland to secrete molting hormone.
In summary, different populations of endocrine cells in insects per-
form different tasks related to growth and development. In general,
populations of cells in multicellular organisms perform different tasks,
and these populations can have profound effects on the entire organ-
ism. Endocrine systems contain glands that secrete hormones, which
are chemical messengers important in cell-to-cell communication. Hor-
mones transfer information from one population of cells to other cells.
Cell populations in multicellular organisms coordinate their functions for
the survival, growth, and reproductive functions of the organism. In the
case of molting hormone, for example, a population of cells in the pro-
thoracic glands produces and secretes the molting hormone after receiv-
ing a signal from a chemical secreted by neurosecretory cells in the brain.
Molting hormone then communicates and directs the activities of a wide
26 CELLS IN TISSUES

variety of other cells. Some populations of cells have important develop-


mental functions, as was explored in this chapter. In some circumstances
these functions may lead to the development of cancer. In the next chap-
ter, the relationship between stem cells, which also have developmental
functions, to cancer cells will be explored.

Bibliography
Wigglesworth VB: The physiology of ecdysis in Rhodnius prolixus
(Hemiptera). II. factors controlling moulting and metamorphosis, Q
J Microsc Sci s2-77:191222, 1934.
Wigglesworth VB: The function of the corpus allatum in the growth
and reproduction of Rhodnius prolixus (Hemiptera), Q J Microsc Sci
79:91121, 1936.
Wigglesworth VB: The determination of characters at metamorphosis in
Rhodnius prolixus (Hemiptera), J Exp Biol 17:201223, 1940.
Wigglesworth VB: The thoracic gland in Rhodnius prolixus (Hemiptera)
and its role in moulting, J Exp Biol 29:561570, 1952.
CHAPTER 3

Similarities and Differences


Between Stem Cells and
Cancer Cells

In the course of development, stem cells are important populations of


cells. Stem cells are cells that have the ability to perpetuate themselves and
to generate mature cells of a particular tissue. Each type of stem cell dif-
ferentiates, or develop, into particular populations of mature cells. For
instance, one population of stem cells is involved in the formation of blood
components, including red and white blood cells. A population of stem
cells exists to continually produce blood cells, which have to be replenished
constantly. They must also be present throughout an adults life.
Stem cells are undifferentiated until they mature into a specific type
of differentiated cell, which cannot divide. Cells in multicellular organ-
isms originate from stem cells, including cancer cells growing in tumors.
Cancer cells are abnormal cells growing in a tumor, which is a growth of
tissue that possesses no physiological function and arises from uncon-
trolled, often rapid cellular proliferation. The loss of the ability to differ-
entiate fully produces cancer cells, which never lose their ability to divide.
This common ability to continue to divide might lead to the hypothesis
that there are similarities between stem cells and cancer cells. Understand-
ing those similarities can lead to strategies for curing or preventing cancer.
These two types of cells will be examined to determine their similarities
and what cellular machinery controls cell growth.
Stem cells are involved in embryonic development, and populations
of stem cells persist throughout adult life. For instance, hematopoietic
stem cells (HSCs) reside in the bone marrow and are involved in produc-
tion of blood cells. Other stem cell populations are involved in producing
28 CELLS IN TISSUES

other differentiated cells, including gut cells, skin cells, and neural cells.
Stem cells may go through particular developmental stages. They can
divide and produce more undifferentiated stem cells or they can divide
into tissue-specific stem cells. From there, they would divide into short-
term progenitor cells and then finally into the differentiated cells.
Populations of stem cells are self-renewing. Both the stem cells and
tissue-specific stem cells have the ability to produce more individuals of
that type. Self-renewal is a critical property of stem cells. If all stem cells
differentiated into other types of cells, there would be no stem cells left
to produce mature cells later in life. Once cells become progenitors, they
are incapable of self-renewal; they cannot produce more of themselves.
Second, stem cells have potential to become a wide variety of other cells.
That is, they are multipotent. However, once a stem cell develops into
a tissue-specific stem cell, the new tissue-specific stem cell cannot then
produce or revert to a multipotent stem cell.
How do stem cells know when to divide? A variety of substances regu-
late the cell cycle (Figure 9). The cell cycle has several phases; the phases
and sequence depicted in Figure 9 are the gap phase to DNA synthesis
phase, which then leads to mitosis. In the absence of appropriate signals,
cells may enter a quiescent stage where they are not in the cycle at all.
The proper signals bring the cell into the gap phase, and once it is past
the restriction point, the cell is committed to continuing the cycle. The
presence of growth factors, such as a transcription factor E2F, takes a cell
past the restriction point; otherwise development remains arrested. The
scheme in Figure 9 is highly simplified to illustrate the basic ways in which
the cell cycle is regulated. Proteins may either inhibit or stimulate other
proteins or their expression or the cell cycle. The dark grey oval protein
above the cell cycle bar inhibits the cell cycle unless it is phosphorylated
twice, represented by the small grey circles attached to the oval protein.
Of course, stem cells and tissue-specific stem cells have much more
complex interactions, so it does not represent all pathways in other types
of stem cells. There is a complexity and variety of stimulatory and inhibi-
tory effects within a typical stem cell.
A stimulatory effect could occur through increased expression of a
gene or by effecting a change in a protein, whereas inhibition could occur
through repression of transcription or binding to a protein. Stimulation
STEM CELLS AND CANCER CELLS 29

supression

protein
activation complex

tran. factor E2F


cell gap phase synthesis
cycle
restriction point
quiescence

Figure 9 Partial schematic cartoon of cell cycle


regulation in adult stem cells. Different proteins
are indicated by different ovals and small circles
are phosphate ions. Arrows in the figure indicate
positive stimulatory effects, and lines with dead ends
represent inhibitory effects. The bar at the bottom
represents phases of the cell cycle.
Source: Modified from He et al., 2009, Figure 2c.

and inhibition is often effected by two classes of genes and their proteins.
Proto-oncogenes code for proteins that regulate cell growth and dif-
ferentiation but can cause cancer when mutated or expression increases.
Tumor suppressors are genes or proteins that protect cells from cancer;
when mutated, they can cause cells to progress to cancer.
There are several pathways of interaction that control whether or not
DNA synthesis commences in stem cells. Once DNA synthesis commences,
the cell is on the path to mitosis, which in a stem cell leads to either dif-
ferentiation or self-renewal. The red symbol at the top of the cell represents
a chemical, often called a mitogen, a chemical substance that activates or
promotes cell division, bound to a membrane protein. That chemical is a
signal to the stem cell, and the binding of the chemical interactions among
proteins inside the cell. Mitogens stimulate both the cyclin D:cyclin-dependent
kinase 4 protein complex and phosphoinositide 3-kinase (PI3K).
In the absence of a mitogen, there are other gene products that con-
trol self-renewal of stem cells. Data on how the interactions in these
30 CELLS IN TISSUES

pathways have been determined in stem cells and whether they affect
self-renewal will be examined next. Components at or near the beginning
of the pathway are good places to start the investigation. Sean Morrison
and his colleagues studied the proto-oncogene Bmi-1. They bred colonies
of mice that were normal, heterozygous for normal and mutated Bmi-1,
and homozygous for the mutated Bmi-1. Morrison and his colleagues
hypothesized that loss of Bmi-1 would affect self-renewal of neural stem
cells. At three different stages of development, 14.5 day old embryos, at
birth, and 30 days after birth, the researchers sacrificed mice and exam-
ined specific areas of their brains (the central nervous system [CNS])
and tissues from the peripheral nervous system (PNS). The central ner-
vous system (CNS) is the brain and spinal cord that coordinates activity
of animals with bilateral symmetry, while the peripheral nervous system
(PNS) connects the limbs and organs to the CNS.
The scientists suspended cells from these nervous system tissues in a
liquid medium. They counted cells using a special cell sorter where cells
are labeled with fluorescent dyes. The dyes are attached to antibodies that
bind to particular cell types. The cell sorter detects and counts the cell
types. The cells were then plated into cell cultures at a low density so
that individual cells, if they grew into populations, formed distinct colo-
nies on the plates. They then counted the number of cells per colony to
determine proliferation and differentiation of individual cells. Regardless
of stage of development, they found similar trends in response, which
was that normal mice always had significantly more cells per colony than
Bmi-1 deficient mice.
Morrison and colleagues also determined the number of cells dying
in each culture and the percentage of 5-bromodeoxyuridine (BrdU) taken
up by each culture. BrdU is a synthetic DNA base that is an analogue of
thymidine and is used to detect cell proliferation. BrdU is incorporated
into newly synthesized DNA of replicating cells (the S phase), substituting
for thymidine during DNA replication. There were no differences in cell
mortality in comparisons of cells from the same type of mouse stem cell
(whether embryonic, newborn, or adult). However, BrdU incorporation
was always much higher in normal mice than in Bmi-1 deficient mice.
In culture, stem cells typically form spheres, making them easy to identify,
and the scientists measured the ability of these new cells to self-renew when
STEM CELLS AND CANCER CELLS 31

Table 7 Effect of loss of Bmi-1 on self-renewal of CNS and PNS


stem cells. Stem cells originated in embryos, newborns, or adults
and were grown for 10 days in culture. Values are the mean number
of new cells formed 1 SD for 3 to 6 independent experiments.
*indicates that the Bmi-1 deficient values were significantly different
from those obtained from normal mice. A, CNS stem cells. B, PNS
stem cells.

A: CNS stem cells normal Bmi-1 mutant


embryo 235 234 7 10*
newborn 164 130 2 3*
adult 202 230 2 3*
B: PNS stem cells normal Bmi-1 mutant
embryo 1650 265 2.6 4*
newborn 540 175 7 5*
adult 396 117 0.1 0.1*

Source: From Molofsky et al., 2003, Figure 1.

transferred to a new culture (Table 7). The numbers shown are the average
number of new stem cells generated from one transferred spherical stem cell.
The data show that stem cells self-renew and proliferate in the pres-
ence of functioning Bmi-1 gene. When the gene is not functioning the
gene product, Bmi-1, is absent from the stem cells; self-renewal, prolifera-
tion of colonies, and DNA synthesis are all drastically reduced in com-
parison to stem cells with Bmi-1. If stem cell self-renewal was not affected
but mortality was higher in the absence of Bmi-1, the scientists might
still have obtained the patterns they found. That is why it was important
to determine the percent mortality in the presence and absence of the
Bmi-1 protein. The results show that mortality was unaffected by the
loss of the gene and its protein, leading to the conclusion that it was self-
renewal and proliferation, not death of existing cells, that were altered.
It turns out that the protein Bmi-1 acts to suppress expression of the
genes p16Ink4a and p19Arf. Both of these proteins are tumor suppressor
proteins and, when functioning, normally prevent phosphorylation of
retinoblastoma (Rb). Note that Bmi-1 is not the only protein that sup-
presses expression of p16Ink4a and p19Arf. Hmga2 also suppresses them,
which helps explain why loss of Bmi-1 did not completely prevent self-
renewal. However, the absence of Bmi-1 should have predictable effects
32 CELLS IN TISSUES

on expression of p16Ink4a and p19Arf, and the consequent effects of loss of


Bmi-1 on self-renewal have already been shown. Morrison and his col-
leagues quantified the expression of p16Ink4a and p19Arf in normal and
Bmi-1-deficient mice.
They performed these measurements for mice at all three stages of
development, using stem cells that had been allowed to grow in culture
for 10 days. The scientists analyzed the ratio of Bmi-1-deficient mice to
normal mice. If the value was more than 1, then Bmi-1-deficient mice
were producing more p16Ink4a or p19Arf. p16Ink4a ratios were all signifi-
cantly higher than one, whether stem cells originated from the CNS,
the PNS, or any stage of development. Expression of p16Ink4a increased
dramatically in adult CNS stem cells of Bmi-1-deficient mice, as well as
newborn and adult PNS stem cells. The ratios ranged from 5.3 to 21.1,
meaning that in the latter case there was 21.1 times as much p16Ink4a
expression in the Bmi-1 deficient mice than in normal mice. Expres-
sion of p19Arf was upregulated to a far lesser extent than p16Ink4a. While
the ratios for p19Arf were not as high, there were all greater than one
(1.4 to 3.0). Because Bmi-1 suppresses expression of p16Ink4a and p19Arf,
its loss allows both proteins to be produced in much higher quantities
than would otherwise be normal, at least at some developmental stages.
This suggests that Bmi-1 is required, at least in the nervous system, to
repress p16Ink4a and p19Arf.
The scientists investigated the loss of the CDKN2 gene (which actu-
ally contains both p16Ink4a and p19Arf genes, with one in an alternate
reading frame from the other) to determine the effects on neural stem cell
self-renewal. They used a strain of mice that were genetically engineered
to not produce p16Ink4a but still retained the ability to produce 19Arf. The
alteration they created in the CDKN2 gene was in the translation start
site for p16Ink4a, which affected that gene only and not the p19Arf, which
started elsewhere and has an alternate reading frame. The scientists per-
formed experiments similar to the ones performed on the Bmi-1-deficient
mice. As before, they measured self-renewal of stem cells, growth of stem
cell colonies in culture, and BrdU incorporation.
Morrison and his colleagues determined that mice deficient in the
p16Ink4a gene had much higher levels of self-renewal, cultured stem cell
proliferation, and DNA synthesis, as measured by BrdU incorporation.
STEM CELLS AND CANCER CELLS 33

p16Ink4a and p19Arf both act, directly or indirectly, to suppress proteins


that phosphorylate retinoblastoma (Rb) protein. Rb strongly binds to the
protein E2F, which is a transcription factor. With one phosphate group
attached, Rb does not bind as strongly to E2F; and with two phosphate
groups attached, Rb no longer suppresses activity of E2F. E2F is part of a
group of proteins that play a role in the cell cycle and are important in the
gap phase to DNA synthesis transition. In the absence of Rb binding, E2F
activates genes that encode proteins involved in DNA synthesis, moving
the cell into the synthesis phase of the cell cycle. So the loss of p16Ink4a leads
to phosphorylation of Rb, which allows E2F to perform its function as a
transcription factor. The ultimate effect of this is cell proliferation.
Morrison and his colleagues next crossbred mice to produce mice
deficient in both the Bmi-1 and the CDKN2 (specifically the p16Ink4a)
genes, and they were thus deficient in both Bmi-1 and p16Ink4a. By
performing various crosses, they were able to study mice with six com-
binations of normal and nonfunctioning Bmi-1 and the CDKN2 genes
(Table 8). They examined self-renewal properties in newborn mice.
In mice that lost function of both Bmi-1 and p16Ink4a, self-renewal was
higher than for mice that lost only Bmi-1. This experiment has the potential
to directly determine whether Bmi-1 promotes neural stem cell self-renewal
by suppressing expression of p16Ink4a. Higher self-renewal in mice that lost
both genes, as compared to mice that lost only Bmi-1, is consistent with
the result that p16Ink4a expression increases in Bmi-1 deficient mice, which
leads to strong suppression of Rb phosphorylation. Then in mice that lost
both Bmi-1 and p16Ink4a, the suppression of Rb phosphorylation is less-
ened, allowing at least partial recovery of the cell cycle and self-renewal. The

Table 8 Genetic make-up of mice used in experiments to test


effects of Bmi-1 and CDKN2 genes on neural stem cells.
strain Bmi-1 genotype CDKN2 genotype
Bmi-1+ /+ and p16 + /+ homozygous normal homozygous normal
Bmi-1+ /+ and p16 + / homozygous normal heterozygous
+ /+ /
Bmi-1 and p16 homozygous normal homozygous deficient
Bmi-1 / and p16 + /+ homozygous deficient homozygous normal
Bmi-1 / and p16 + / homozygous deficient heterozygous
Bmi-1 / and p16 / homozygous deficient homozygous deficient

Source: From Molofsky et al., 2003.


34 CELLS IN TISSUES

recovery is partial because p19Arf is also involved in suppressing the cell cycle
through several intermediary proteins.
The components Bmi-1 and p16Ink4a are at or near the beginning of
the pathway. Studying the impact of their absence is a clever technique to
confirm their role in the cell cycle and stem cell self-renewal, and that is
exactly what Morrison and his colleagues did. There are other pathways
involved in regulating stem cell self-renewal, but this example points out
the complex controls on stem cell self-renewal.
Controls on self-renewal maintain populations of cells. Stem cells
have the ability for self-renewal, but self-renewal in many tissue-specific
stem cell populations is controlled by the cell cycle. Differentiation of
stem cells requires another set of complex controls. For any tissue that fre-
quently becomes damaged or is exposed to a high degree of wear and tear,
it would be expected to possess an active population of stem cells capable
of producing the cells that make up that tissue. Certainly blood, skin, and
the cells that line the alimentary canal would fit that category. Lung cells
are exposed to the external environment every time a person inhales, and
they are frequently exposed to pollutants in the air. Long-term exposure
to pollutants causes wear and tear on the tissues of the lungs.
Lung stem cell populations would be important in generating new
cells to replace damaged ones, and the controls on self-renewal and dif-
ferentiation should be similar to the controls in other stem cell popula-
tions. Lung cancer is a common and often fatal form of cancer. Is there
any relationship between the regulation of stem cell self-renewal and
cancer?
The genes and proteins involved in controlling the cell cycle were earlier
called proto-oncogenes and tumor suppressor proteins. With names like these,
there is likely a known relationship between these genes and proteins and the
formation of cancer. Daniel Betticher and a team of colleagues studied sev-
eral of the cell cycle genes and in human lung cancer. There are several dif-
ferent types of cancer that can develop in lungs. Betticher and his colleagues
studied non-small cell lung carcinoma (NSCLC; carcinoma is another term
for cancer), which makes up about 80% of all lung cancers. The name dif-
ferentiates it from small cell lung carcinoma (SCLC). Compared to SCLC,
NSCLCs are relatively insensitive to chemotherapy, which is why it is
important to understand how these cancers develop and grow. Their goal
was to examine lung cancers for expression of proteins involved in the cell
STEM CELLS AND CANCER CELLS 35

cycle, specifically some of the ones discussed earlier with regard to stem cell
self-renewal and the cell cycle: p16Ink4a, p19Arf, p53, and Rb protein.
The scientists obtained tumor samples from 49 patients who had
them removed surgically. The scientists obtained cells from each tumor
and created cultures of them in environmentally-controlled incubators.
In addition, the scientists cultured healthy lung tissue, a human leukemia
cell line (K562) known to have the p16/p19 gene locus (CDKN2) deleted
and two other cell lines known to contain and express CDKN2.
Cells from each culture were harvested and suspended in solution.
From the resulting suspension, they took a standard amount of pro-
tein. The scientists separated the proteins using gel electrophoresis. The
proteins were probed with antibodies specific to Rb, p16Ink4a, p53, and
p19Arf. In addition, the scientists also used antibodies to stain whole cells
from all cultures for these proteins. The scientists detected proteins using
a luminescence detection system; antibodies that bound to a particular
protein were luminescent on the gels and in the cells. Their controls were
analyzed to be sure that detection of proteins was accurate. Only three
of 49 tumors expressed normal levels of all four proteins; all others con-
tained abnormal expression (either downregulated, with expression lower
than normal, or overexpressed) of at least one of these proteins (Table 9).

Table 9 Numbers of lung cancer tumors with abnormal


expression of proteins involved in the cell cycle and
self-renewal of stem cells. Altered expression is either
downregulation or overexpression of the protein.
altered expression
percentage of tumors
p19Arf p16Ink4a p53 Rb
6.1 yes yes yes
8.2 yes yes yes
12.2 yes yes
4.1 yes yes
2.0 yes yes
22.4 yes yes
4.1 yes yes
16.3 yes
6.1 yes
6.1 yes
6.1

Source: From Vonlanthen et al., 1998, Table 3


36 CELLS IN TISSUES

No normal lung cell cultures contained detectable levels of p16Ink4a


and p19Arf. The scientists examined the relationships among downregula-
tion or overexpression of the four proteins shown in Table 9 (shown in
Table 10). In any comparison in Table 10, if there is a positive association
between the altered expression of two proteins, there will be more tumors
with both proteins altered than expected by chance. For instance, there is
a positive association between downregulation of p16Ink4a and overexpres-
sion of p53. These two events are more likely to occur together in lung
cancer than expected by chance. If there is a negative association, there
will be fewer tumors where both are altered and more where one is altered
and the other is not than expected by random chance. That is the case for
downregulation of p19Arf and overexpression of p53.
Recall that p16Ink4a and p19Arf are transcribed by the same gene
(CDKN2), but p19Arf is an alternate reading frame. In a subset of the tumors
(28) and in several normal lung samples, the scientists examined the expres-
sion of p16Ink4a and p19Arf by reverse transcriptase polymerase chain reaction
(RT-PCR; Table 11). In RT-PCR, mRNA strands are reverse transcribed into
their DNA complement (complementary DNA, or cDNA), and the result-
ing cDNA is amplified and analyzed for presence and quantity.

Table 10 Relationships among altered expression


of some of the cell cycle proteins in lung cancer
tumors. NS - Not statistically significant.
Rb p-value
altered normal
altered 3 21
p16Ink4a 0.003
normal 12 10
p53
altered normal
altered 15 8
p16Ink4a 0.015
normal 6 17
p53
altered normal
altered 5 14
p19Arf 0.025
normal 16 11
p19Arf
altered normal
altered 13 11
p16Ink4a 0.08 (NS)
normal 6 16
Source: From Vonlanthen et al., 1998.
STEM CELLS AND CANCER CELLS 37

Table 11 Expression of p16Ink4a and p19Arf transcripts in


normal lung tissue and lung cancer tumors.
category normal lung tumor
equal levels of p16Ink4a and p19Arf mRNA 6/6 16/28
p19Arf mRNA detected 6/6 28/28
p16Ink4a mRNA detected 6/6 16/28
both proteins strongly expressed 11/28
both proteins absent 0/6 5/28

Source: From Vonlanthen et al., 1998.

At least one of the four cell cycle proteins was altered in 93.5% of the
tumors (see Table 9). Twenty-two had at least two of the genes, CDKN2
(p16Ink4a/p19Arf) and/or p53 altered, and for those 22, Rb expression was
normal. Normal lung tissue expressed both CDKN2 transcripts equally,
whereas only 57% of tumors expressed both transcripts equally (see
Table 11). That lower percentage was due to altered transcription of p16Ink4a.
Alterations in expression could occur from mutations in the gene or altered
signals that affect the expression of a gene that produces a particular pro-
tein product. The signal could turn on, off, up, or down the expression of a
gene. Recall in stem cells that loss of Bmi-1 reduced stem cell self-renewal,
and loss of p16Ink4a increased self-renewal. If p16Ink4a is downregulated or
mutated, it could cause unregulated growth. Cancer cells display unregu-
lated growth. Downregulation of both p16Ink4a and p19Arf should both
lead to relaxation of the cyclin complex suppression, which would lead to
phosphorylation of Rb. Once phosphorylated, Rb no longer suppresses
E2F, and this can take the cell into the synthesis phase of the cell cycle.
Altered expression of p53 and p16Ink4a both separately lead to effects
on Rb phosphorylation; the effect of alteration of both could be larger
than the effect of either alone, an example of an emergent property.
The two pathways involving p16Ink4a and p19Arf/p53 could cooperate in
tumor growth inhibition; and when either or both is altered, cells may
enter the cell cycle unchecked and become cancerous. Betticher and his
colleagues found that some lung cancer tumors with p16Ink4a alterations
also had p53 mutations. In lung cancers, both p16Ink4a and p19Arf are
frequently altered, and the scientists found a linkage between p19Arf and
p53. The negative association between p19Arf downregulation and abnor-
mal p53 expression suggests a common pathway in malignant growth and
38 CELLS IN TISSUES

supports the hypothesis that the p19Arf peptide plays a role in human can-
cer. The scientists concluded loss of two controls on the cell cycle, p16Ink4a
downregulation and aberrant p53 expression, leads to a high probability
of NSCLC tumor formation. Mutations in several of the genes involved
in the cell cycle are also known to play a role in a variety of other cancers.
Although some of the controls on the cell cycle in stem cells have been
discussed, it is important to determine whether these controls, or altera-
tions in them, play a role in developing cancer. In lung cancer, and pos-
sibly other cancers, alteration of the CDKN2 locus can simultaneously
impair both the p16Ink4a- cyclinD/CDK4-Rb and the p19Arf-p53-Cip/
Kip-cyclinE/CDK2-Rb pathways. When DNA mutates or cells fail to func-
tion normally for some other reason, tumors may begin to grow, depend-
ing upon other changes that have occurred. The connection between stem
cells and cancer cells is that the biochemical machinery that regulates self-
renewal and the cell cycle in stem cells contains many proteins that, when
altered, lead to unregulated growth. Cancer is a group of diseases char-
acterized by uncontrolled growth and spread of abnormal cells. If cancer
is considered as a disease of unregulated self-renewal, the connection to
regulation of stem cell self-renewal is clear. Tumors may originate from the
transformation of signaling pathways in normal stem cells.
Each cell type in a multicellular organism exists as a population of
cells, and each has a particular structure related to its function. Over
evolutionary time, populations of cells within multicellular animals have
evolved complex mechanisms of regulation, which can become dysfunc-
tional through mutation or improper signaling. Stem cells have no regular
day-to-day function and remain undifferentiated. They await signals to
proliferate and differentiate. Cancer cells have properties similar to stem
cells. In cancer cells, the proliferation signal may always be on; although
some types of cancers grow faster than others, the unregulated or dysfunc-
tional cell cycle is common.

Ethical, Legal, Social Implications: Decisions About to


Whom and When Medical Interventions are Given
Sickle-cell disease, malaria, amyotrophic lateral sclerosis (ALS), Lyme dis-
ease, and cancer have all been discussed in this book and another book
STEM CELLS AND CANCER CELLS 39

in this series, Effects of Genetic and Pathogenic Diseases on Cells. Modern


medicine and our ability to keep people alive with debilitating or deadly
diseases raise ethical and legal issues. The historic case of Terri Schiavo
brought national attention to the issue of medical intervention, a course
of action designed to improve health or alter the course of disease. Schiavo
was 26-years-old when she collapsed in her home with no pulse or respira-
tion. She was resuscitated, but the long period without oxygen led to brain
injury, and ultimately she was diagnosed as being in a persistent vegetative
state (PVS). After many years in court, her husband won his case for her
to be disconnected from her life-sustaining feeding tube, which resulted
in her death. The battle in court centered on her husband, who argued
for removal of the feeding tube, and her parents, who argued that she was
conscious. The court ultimately ruled that Terri Schiavo would not wish to
continue life-prolonging measures, but not before state and federal politi-
cians, pro-life, and disability rights advocacy groups became involved. She
lived in a PVS for 15 years, surviving because of the feeding tube, a medical
intervention. Disconnecting her from this intervention led to her death.
Both sides in the case presented an ethical argument. Schiavos hus-
band argued, in part, that she should not be forced to live a life in a PVS.
Further, he argued that she would choose to not continue living in that
state with no hope of recovery or return to normal biological function.
The ethical thing to do would be to allow her to die. Her parents argued
that she was conscious and was aware of her surroundings. It would not
be ethical to remove the feeding tube and cause her to die.
Advances in medical science and technology have made many inter-
ventions possible than were conceivable even several decades ago. So
who decides what medical interventions to give and when to give them?
There are cases where medical interventions are requested by patients or
prescribed by physicians for patients that are aware and can make deci-
sions about their own course of treatment. Humans have a tendency to
accept any medical intervention that may improve their personal health
or increase chances for survival or even societal status. Interventions range
from plastic surgeries to heart and brain operations with the potential for
gene therapy in the future.
Individuals may opt for plastic surgeries to maintain a youthful
appearance or to alter their physical appearance. These elective surgeries
40 CELLS IN TISSUES

are clearly requested by the patient, and may not directly affect health or
survival of the patient. However, if the surgery increases the individuals
self-esteem and attitude, then there may be an indirect benefit to their
health. More immediate emergencies may require rapid and extreme
interventions to keep a patient alive, such as when a patient has a heart
attack, stroke, or brain embolism.
Some interventions may be preventative, designed to stave off future
problems. Intervening after the fact may be easier for patients and physi-
cians. Consider an obese patient who has been told by his doctor to either
lose weight, or face heart disease and diabetes later in life. The patient
refuses to comply with a change in lifestyle, which would be a medical
intervention, but readily accepts insulin and heart medication after he
has developed diabetes and heart disease. So some types of interventions
may be more readily accepted, such as when there is a life-threatening
emergency or when a prescription drug can be used in place of a change
in lifestyle.
Other interventions (such as, organ transplants) are not so easy, and
healthy organs for transplant may be in short supply. For organ trans-
plants there are specific principles that the medical community adheres
to in order to most ethically share the scarce resource of healthy organs.
Principles used to decide on the use of these sorts of medical interven-
tions can be categorized according to several fundamental values: treat-
ing people equally, favoring the worst-off, maximizing total benefits, and
promoting and rewarding social usefulness.
Equal treatment may be accomplished through a lottery or through a
first-come, first-served principle. The worst-off patients may be the sickest
or the youngest. Those who are sickest are currently suffering; those who
are youngest may be prioritized because they have the most years in front
of them. When maximizing total benefit, principles may be used to save
the most lives or those patients with the best prognosis for benefitting
from the intervention. Finally, promoting and rewarding social usefulness
provides priority to those who are in the best position to help society in
the future or have greatly helped society in the past and have suffered for it.
According to some ethicists, some of these principles are flawed, in
that they recognize ethically irrelevant considerations. Other principles
STEM CELLS AND CANCER CELLS 41

are insufficient, in that they ignore other ethical considerations. The


insufficient principles ought to be integrated into a comprehensive
method of making ethical decisions about medical interventions, and
the flawed principles must be eliminated from the decision-making pro-
cess. For instance, first-come, first-served is flawed because individuals
who arrive first may not be the most deserving of treatment under other
ethical considerations. Prioritizing the sickest individuals first is also
flawed, whereas curing the youngest first is not.
In deciding the case of Terri Schiavo, the medical community primar-
ily applied the sickest first and first-come, first-served principles, neither
of which is recommended by some ethicists. Because Schiavo arrived at
the hospital before others, and the hospital only has a certain number of
feeding tubes and beds, her arrival may have precluded someone else from
obtaining proper treatment. Schiavo was relatively young, in her 20s, but
she did not have a favorable prognosis.
The complete lives system of medical intervention decision-making
combines five principles: (1) the youngest-first, (2) prognosis, (3) save the
most lives, (4) lottery, and (5) instrumental value (especially of healthcare
workers during public health emergencies). It has the advantage that it
incorporates a large number of ethical principles, but this system may not
work well for decisions about allocating medical resources outside of the
organ transplant system. In addition, integrating these principles may be
difficult; lottery may lead to selection of an elderly patient with a poor
prognosis.

Bibliography
He S, Nakada D, Morrison SJ: Mechanisms of stem cell self-renewal,
Annu Rev Cell Dev Biol 25:377406, 2009.
Hook CC, Mueller PS: The Terri Schiavo saga: the making of a tragedy
and lessons learned, Mayo Clin Proc 80(11):14491460, 2005.
Persad G, Wertheimer A, Emanuel EJ: Principles for allocation of scarce
medical interventions, Lancet 373(9661):423431, 2009.
Reya T, Morrison SJ, Clarke MF, et al: Stem cells, cancer, and cancer stem
cells, Nature 414(6859):105111, 2001.
42 CELLS IN TISSUES

Molofsky AV, Pardal R, Iwashita T, et al.: Bmi-1 dependence distinguishes


neural stem cell self-renewal from progenitor proliferation, Nature
425:962967, 2003.
Taipale J, Beachy PA: The hedgehog and Wnt signalling pathways in cancer,
Nature 411:349354, 2001.
Vonlanthen S, Heighway J, Tschan MP, et al.: Expression of p16INK4a/
p16alpha and p19ARF/p16beta is frequently altered in non-small
cell lung cancer and correlates with p53 overexpression, Oncogene
17(21):27792785, 1998.
Conclusion
Cells divide, and that leads to population growth even in multicellular
organisms. Cell division and population growth illustrates how all cells
come from preexisting cells. Cell division is highly regulated through the
mechanisms of the cell cycle. The high degree of regulation of the cycle
is part of the maintenance of internal environments, which differ from
external environments of cells. Communication between members of the
same or different population of cells is part of this regulation, as discussed
in the chapters on how the digestive and endocrine systems work. There
are advantages and costs to being multicellular, but the evolution of mul-
ticellularity suggest that the costs outweigh the benefits. Division of labor
among population of cells in tissues or organs is necessary for multicel-
lular organisms to function and survive.
Glossary
alimentary canal. The alimentary canal is long tube in the body through which
food passes after it is ingested.
antagonist. A chemical that prevents the physiological effects of another chemi-
cal, such as epinephrine.
assimilation. Assimilation is the process of absorbing nutrients and incorporating
them into the body.
cancer cells. Cancer cells are abnormal cells growing in a tumor.
capillaries. Capillaries are small blood vessels that form a network between arte-
rioles and venules in the circulatory system.
carcinoma. A cancerous tumor derived from epithelial tissue.
cells. The smallest structural and functional unit of an organism.
central nervous system. The central nervous system (CNS) is the brain and
spinal cord that coordinates activity of animals with bilateral symmetry.
connective tissue. Tissue that connects, supports, separates, and helps hold
together other tissues and organs.
corpus allatum. An endocrine gland in insects and related invertebrates that
secretes juvenile hormone.
differentiates. In cell biology, the process of a cell changing from one cell type
to another.
digestion. Digestion is the process of breaking down food.
endocrine system. An organ system in many animals that is made up of glands
that secrete hormones.
epidermis. An epidermis is an outer layer of cells in multicellular organisms.
Epithelial cells. Epithelial cells make up the epithelium that encloses and pro-
tects organs and internal surfaces that have direct contact with the external
environment.
epithelium. Thin tissue that forms the outer layer of a bodys surface and lines
hollow structures such as the alimentary canal.
esophagus. Muscular tube in the digestive system through which food passes
from the throat to the stomach.
evolved. To have undergone evolution and changed gradually.
exoskeleton. An exoskeleton is a hardened external supportive covering com-
posed of nitrogen-containing polysaccharides, protein, and lipids.
feces. Waste material discharged from the bowels; undigested, unassimilated
food.
gallbladder. The small sac-shaped organ beneath the liver that stores bile.
ganglia. Ganglia are masses of nerve tissue containing cell bodies of neurons.
46 GLOSSARY

gastric pits. Indentations in the stomach that mark entrances to the gastric glands.
hemolymph. Hemolymph is the body fluid of insects.
histamine. A chemical released by cells in response to injury and in allergic and
inflammatory reactions, causing contraction of smooth muscle, dilation of capil-
laries, and known to activate parietal cells in the stomach.
homeostasis. maintain internal conditions within a range of acceptable extremes.
hormones. Hormones are chemical substances, usually peptides or steroids,
produced by one cell type, that control and regulate activity of other cells.
information. Information is stored, communicated, and implemented or
interpreted.
ingestion. Ingestion is the process of taking food into the body.
juvenile hormone. An insect hormone that regulates larval development and
inhibits metamorphosis.
large intestine. An organ of the digestive system of mammals containing the
cecum, colon, and rectum.
liver. An organ of the digestive system responsible for detoxification, carbohy-
drate metabolism, and other metabolic processes.
lumen. Lumen is the inner open space or cavity of a tubular organ.
lymphatic vessels. Lymphatic vessels are thin walled, valved structures that carry
lymph, a colorless fluid that contains white blood cells.
metamorphosis. Metamorphosis is an abrupt developmental change in the form
or structure of an animal.
mitogen. A mitogen is a chemical substance that activates or promotes cell
division.
molting. The process of shedding a cuticle in insects and other invertebrates with
exoskeletons during development.
mucus. A slimy substance, typically not miscible with water, secreted by mucous
membranes and glands for lubrication and protection.
multicellular. Organism consists of many interdependent cells, which cannot
exist on their own.
multipotent. Multipotent cells have potential to differentiate into any number
of other cells.
muscle cells. An elongated contractile cell that forms muscles.
neurons. The basic cell type of the nervous system of animals that transmits nerve
impulses.
neurosecretory cells. Neurons that function to translate neural signals into chem-
ical signals by secreting neurohormones.
organisms. An individual animal, plant, or single-celled life form.
pancreas. the organ in your abdomen that makes insulin, among other compounds.
parietal cells. Specialized epithelial cells in the gastric glands of mammalian
stomachs that secrete hydrochloric acid.
pepsin. An enzyme that breaks down proteins.
GLOSSARY
47

pepsinogen. A protein secreted by the stomach and converted into the enzyme
pepsin.
peripheral nervous system. The peripheral nervous system (PNS) connects the
limbs and organs to the CNS.
peripheral nervous system (PNS). The peripheral nervous system (PNS) con-
nects the limbs and organs to the CNS.
peristalsis. Muscular contractions that move food through the alimentary canal.
population. A population is a group of individuals of the same species living in
the same place at the same time
prothoracic gland. An endocrine gland in insects and related invertebrates that
secretes molting hormone.
protocerebrum. The first segment of the brain in an insect that innervates the
compound eyes.
proton pump inhibitor (PPI). A chemical that inhibits the activity of proton
pumps.
proto-oncogenes. Genes that code for proteins that regulate cell growth and dif-
ferentiation but can cause cancer when mutated or expression increases.
salivary glands. Glands in the digestive system of animals that secrete saliva.
second messenger. An intracellular chemical that mediates cell activity by relay-
ing a signal from an extracellular molecule bound to the cells surface.
small intestine. An organ of the digestive system of mammals responsible for
much digestion and assimilation of nutrients.
smooth muscle. Smooth muscle is a contractile type of muscle tissue controlled
by the involuntary nervous system, occurring in the walls of the stomach, intes-
tines, and blood vessels.
stem cells. Long lived, produce daughter cells. and replenish themselves indefinitely.
stomach. An organ of the digestive system responsible for some digestion and
temporary storage of food.
tissues. Collections of more than one cell type of a single species that work
together to perform a complex function.
tubulovesicles. Tube-shaped vesicles in parietal cells.
tumor suppressors. Tumor suppressors are genes or proteins that protect cells
from cancer; when mutated, they can cause cells to progress to cancer.
tumors. Abnormal growths of tissue.
undifferentiated. Not differentiated into a particular cell type, as in an undif-
ferentiated stem cell.
Index
Accessory glands, 3 Fats. See Lipids
Alimentary canal, 1, 2 Feces, 15
mammalian digestive 5-bromodeoxyuridine (BrdU), 30
system, 23
Antagonist, 8 Gallbladder, 3
Assimilation, 1, 2 Ganglia, 24
Gastric pits, 56
Betticher, Daniel, 34, 37 Gastrin, 8
Bicarbonate, 10 Glucose absorption and transport, 12
Bmi-1, 3032 Glucose transporter 2 (GLUT2),
effects on neural stem cells, 3334 1215
loss effect on self-renewal of CNS/ Gut. See Alimentary canal
PNS stem cells, 31
Hematopoietic stem cells (HSCs), 27
Cancer cells, 27, 38 Hemolymph, 19
ethical, legal, social implications of, Histamine, 78
3841 Homeostasis, 2
versus stem cells, 2741 Hormones, 17
Capillaries, 12 juvenile, 22, 23
Carbohydrates, 12 molting, 20, 22, 24, 2526
Carcinoma, 34 Human digestive system, anatomy
CDKN2, 32, 33, 35, 36, 38 of, 3
Central nervous system (CNS), 30, 31
Connective tissue, 5 Information, 1
Corpus allatum, 21, 23 Ingestion, 1, 2
Critical period, 19
Cyclic adenosine monophosphate Juvenile hormone, 2224
(cAMP), 89
Large intestine, 2
da Vinci, Leonardo, 2 Lipids, 11
Differentiated cells, 27 Liver, 3
Digestion, 1, 2 Lumen, 5
Luminescence detection system, 35
Endocrine system, 17, 25 Lung cancer, 34, 35, 36, 37
Epidermis, 18 Lymphatic vessels, 12
Epithelial cells, 5
small intestine, 11, 15 Medical intervention decision-
Epithelium, 5 making, ethical, legal, social
Esophagus, 2 implications of, 3841
Exoskeleton, 17 Metamorphosis, 21
molting of, 17, 18, 20 Microvilli, 12
50 INDEX

Mitogen, 29 Reverse transcriptase polymerase


Molting, 20, 22, 24, 2526 chain reaction (RT-PCR), 36
Morrison, Sean, 30, 32, 33 Rhodnius prolixus (R. prolixus), 18,
mRNA, 36 21, 23
Mucosa, 5, 12 brain transplant experiments, 24
Mucus, 10 decapitation experiments, 20, 23
Multicellular organisms, 1 molting of, 20
Multipotent, 28 Rder, Pia, 12
Muscle cells, 1
smooth, 5 Salivary glands, 3
Muscularis externa, 5 Schiavo, Terri, 3941
Schwann, Theodor, 10
Neurons, 1 Second messenger, 8
Neurosecretory cells, 25 Small cell lung carcinoma
Non-small cell lung carcinoma (SCLC), 34
(NSCLC), 34, 38 Small intestine, 2
epithelial cells and glucose
Organ transplantation, 40 transport, 11
Smooth muscle, 5
Pancreas, 3 Sodium-dependent glucose
Parietal cells cotransporter (SGLT1),
activation of, 6 1214
pH changes on, 67 Stem cells, 27, 38
representation at rest and under versus cancer cells, 2741
stimulation, 10 cell cycle regulation in, 2829
Pepsin, 10 multipotent, 28
Pepsinogen, 10 population of, 27
Peripheral nervous system (PNS), tissue-specific, 28
30, 31 Stomach, 2
Peristalsis, 10 layers and cell types of, 5
Persistent vegetative state (PVS), 39 lumen of, 5
Phosphorylate retinoblastoma
(Rb), 33 Tissues, 1
Population, 1 connective, 5
Proteins, 11 Tissue-specific stem cell, 28
Prothoracic gland, 25 Tubulovesicles, 9
Protocerebrum, 25 Tumors, 27
Proton pump inhibitor (PPI), 8 Tumor suppressors, 29, 34
Proto-oncogenes, 29, 34
Prout, William, 4 Wigglesworth, Vincent, 1726
OTHER TITLES IN OUR BIOLOGY
COLLECTION

Behavior and Information Exchangeby Christopher J. Paradise and A. Malcolm Campbell


Ecological Dynamicsby Christopher J. Paradise and A. Malcolm Campbell
Evolution of Interactions in Communitiesby Christopher J. Paradise and
A.Malcolm Campbell
Evolutionary Historyby Christopher J. Paradise and A. Malcolm Campbell
Effects of Genetic and Pathogenic Diseases on Cellsby Christopher J.Paradise and
A.Malcolm Campbell
Information in the Environmentby Christopher J. Paradise and A. Malcolm Campbell
Mechanisms of Evolutionby Christopher J. Paradise and A. Malcolm Campbell
Properties in and of Populationsby Christopher J. Paradise and A. Malcolm Campbell
Variation and Population Geneticsby Christopher J. Paradise and A. Malcolm Campbell
Ecological Homeostasisby Christopher J. Paradise and A. Malcolm Campbell
Ecological Interactionsby Christopher J. Paradise and A. Malcolm Campbell
Emergent Properties of Individual Organismsby Christopher J. Paradise and
A. Malcolm Campbell
Organismal Homeostasisby Christopher J. Paradise and A. Malcolm Campbell
Population Homeostasisby Christopher J. Paradise and A. Malcolm Campbell

Announcing Digital Content Crafted by Librarians


Momentum Press offers digital content as authoritative treatments of advanced engineering
topics by leaders in their field. Hosted on ebrary, MP provides practitioners, researchers, faculty,
and students in engineering, science, and industry with innovative electronic content in sensors
and controls engineering, advanced energy engineering, manufacturing, and materials science.

Momentum Press offers library-friendly terms:


perpetual access for a one-time fee
no subscriptions or access fees required
unlimited concurrent usage permitted
downloadable PDFs provided
free MARC records included
free trials

The Momentum Press digital library is very affordable, with no obligation to buy in future
years.
For more information, please visit www.momentumpress.net/library or to set up a trial in the
US, please contact mpsales@globalepress.com.
EBOOKS Cells in Tissues
FOR THE Christopher J. Paradise A. Malcolm Campbell
APPLIED BIOLOGY COLLECTION
Two systems illustrate how individual cells of an organ system
SCIENCES
function, communicate, and coordinate activities. The digestive
LIBRARY system breaks down and absorbs nutrients, and some special-
Create your own ized cells break down and absorb nutrients. The case of parietal
Customized Content cells in the stomach and epithelial cells in the small intestine

Cells in Tissues
Bundlethe more are used to describe how cells function as a unit within organ
books you buy, systems, coordinating activities and communicating with one
the greater your another. The endocrine system of insects affects molting and
discount! metamorphosis, and specialized cells are also important in each
of these processes within that organ system. The experiments
that were devised to determine the role of hormones in insect
THE CONTENT
molting and metamorphosis are described. Finally, stem cells
Energy Physics are healthy components of several different systems in animal
Engineering bodies and are described in relation to a disruption in function.
Biotechnology In this breakdown of function, cancer cells, in contrast to stem
Biology cells, can abnormally affect cell cycle regulation.
Mathematics
Christopher J. Paradiseis professor of biology and environ-
Chemistry
mental studies at Davidson College. He teaches introductory
biology, ecology, entomology, and topical seminars on ecotoxi-
THE TERMS cology and renewable natural resources. He also occasionally
Perpetual access leads a study abroad program in India. His research evaluates
for a one time fee anthropogenic factors that influence insect biodiversity at a
No subscriptions or variety of scales. His current research interests include effects of
access fees land use patterns on pollinator communities in parks.
Unlimited
A. Malcolm Campbellteaches biology at Davidson College,
concurrent usage
NC. He received national and international education awards:
Downloadable PDFs Genetics Society of America (2013); American Association for the
Free MARC records Advancement of Science (2012); and American Society for Cell
Biology (2006). He was the founding co-editor in chief of CBE Life
For further information,
Sciences Education; founding director of Genome Consortium
a free trial, or to order,
contact: for Active Teaching (GCAT); and member of the American Soci- Christopher J. Paradise
sales@momentumpress.net ety for Cell Biology governing council (20122014).
A. Malcolm Campbell