Download By Allison Byrum / Intern

Ann Richmond's laboratory gains new insights into tumor
growth and wound healing through studies of the "SOS gene
By Allison Byrum / Intern
Jan. 19, 2001
OVERVIEW
You might call MGSA the SOS gene. That’s because it is switched on in the vicinity of a wound
and produces a special protein, called MGSA, that attracts the body’s repair crew—infectionfighting white blood cells and cells involved in the formation of new skin and blood vessels.
In the 1980s, however, when Ann Richmond began studying this lightweight protein, it was in a
much different context. She was searching for a factor that helps melanoma tumors grow. Even
before she and her collaborators had proven that such a protein exists, they had dubbed the
molecule “melanoma growth stimulatory activity,” or MGSA.
As a result of their persistent efforts, Richmond and her colleagues successfully characterized
MGSA and so proved the existence of factors that promote tumor growth. Today, Richmond is a
professor of cancer biology and her laboratory in Vanderbilt’s Medical Center and the Nashville
Veteran’s Affairs Medical Center is still actively pursuing the scientific trail that opened up when
she identified MGSA more than 10 years ago.
Since its discovery, researchers have found that MGSA plays a number of important roles in the
body. It is essential to wound healing and is actively involved in inflammatory diseases such as
rheumatoid arthritis and psoriasis. Since its initial characterization, MGSA has also been linked to
several other types of cancer including lung, breast and other skin cancers. The statistics on
melanoma deaths alone are frightening1. When combined with the death toll of the other cancers
in which MGSA is implicated2, the resulting figures dramatize just how great the potential payoff
from a better understanding of MGSA’s activities can be.
If that weren’t enough, further investigation has shown that MGSA is a member of a larger group
of proteins, called chemokines3, that play a number of critical roles. Close to 100 chemokines
have now been found. Some, like MGSA, recruit white blood cells to wounds and act as growth
factors for many cancers. But others aid in the formation of the immune system during the
embryonic development and some have been found that even counteract tumor growth.
Scientists now know that MGSA is found in all animals and is one of the keys to wound healing.
Any injury, for example a cut on an arm or blistering sunburn, causes cells near the wound to
send out SOS messages to the surrounding tissues. This message is sent in the form of MGSA,
which travels into the cells around the wound and attracts white blood cells, new skin cells, and
1
Melanoma is the most serious form of skin cancer and accounts for 79% of skin cancer deaths. According
to the American Cancer Society, approximately 47,700 new cases of melanoma were reported in the year
2000 and the disease claimed the lives of approximately 7,700 people. Melanoma is one the most common
cancers to effect people under the age of 30, although more than half of all melanoma cases occur in people
over the age of 50.
2 Breast, lung, and other skin cancers combined with melanoma claimed the lives of approximately 2.5
million people in 2000. Lung cancer is the leading cause of cancer death in the US, while skin and breast
cancer are the two most common cancers in women.
3 Chemokines are small proteins that cause cells to move. Since the characterization of MGSA, a huge
family of chemokines has been discovered. Now that so many have been found, scientists have group them
into different families. MGSA’s “family name” is CXC and it has been given a second name CXCL1.
-1-
Ann Richmond's laboratory gains new insights into tumor growth and
wound healing through studies of the "SOS gene
OVERVIEW
cells required to form new blood vessels to the damaged site. Once this repair team is
assembled, the SOS message is cut off. Help has arrived and no more MGSA is needed.
Problems can arise, however, when the SOS message does not turn off normally. When MGSA is
continuously produced, the body continues to respond long after the appropriate help has arrived.
In certain circumstances, this can give rise to tumors. In addition, established tumors produce
MGSA in order to establish connections to nearby blood vessels. “In many ways, tumors are like
never-healing wounds,” Richmond says.4
Before Richmond characterized MGSA, little was known about this class of proteins. A
postdoctoral fellow at Emory University at the time, she hypothesized that melanoma tumor cells
must be producing something that promotes their growth. She and Dave Lawson 5, who was an
oncologist at Emory, set out to purify the protein that they guessed was helping tumors grow.
The research was not easy. In order to get samples of melanoma to experiment with, Richmond
and Lawson had to visit melanoma patients in the hospital to get their informed consent.
Richmond describes this as one of the most trying experiences in her early research. Day after
day she would sit at the bedsides of patients at different stages of the disease and listen to their
stories, their hopes and their dreams. She still remembers many of the patients vividly, most of
whom did not survive their illness.
Nevertheless, “these experiences lit a flame of work in us,” Richmond remembers. “We had to
continue for each of these people.”
In addition to the emotional strains specific to the work, Richmond’s team had to cope with
insufficient resources. The tiny lab they were given for their experiments barely accommodated
their equipment, much less the researchers. That first summer “we only hired skinny people!”
Richmond recalls, laughing. Space problems were compounded by the fact that the team did not
have the funds they needed to buy new equipment. So they were forced to work late nights using
borrowed equipment, which broke down all too frequently because of the unusual demands they
were putting on it.
Finally, however, grants came through and the research began moving. After seven years of
research, a few blind alleys and many sleepless nights, Richmond and post-doctoral fellow Greg
Thomas6 managed to purify and characterize MGSA.
Midway through their characterization effort, the team discovered that the protein they were
calling MGSA looked very similar to another already characterized protein: platelet factor four.
Platelet factor four is a common factor found in the blood stream. Although the two proteins
weren’t exactly the same, they were similar enough that Richmond became fearful that her
protein was simply another version of the familiar factor. That caused her to seriously consider
abandoning the attempt to complete her characterization of MGSA.
Influenced by the counsel of a colleague 7, she decided to continue the effort. That was fortunate
because not only did MGSA turn out to perform a completely different function than platelet factor
four, but also the similarity that had alarmed Richmond initially proved to be significant: It opened
4
Angiogenesis is the outgrowth of tiny new capillaries from existing blood vessels. It is important in wound
healing because new blood vessels must grow into the site of a wound to help it heal. It also contributes to
tumor growth, since the new blood vessels feed the tumor.
5 Now associate professor of medicine at Emory University’s Winship Cancer Institute.
6 Later became department director at Solvay Pharmaceuticals in Atlanta.
7 Skip Catherwood, a research colleague at the Veterans Affairs Medical Center in Atlanta, Georgia, advised
Richmond to follow through on the characterization. He advised Richmond to “play out the cards she had
been dealt.”
-2-
Ann Richmond's laboratory gains new insights into tumor growth and
wound healing through studies of the "SOS gene
OVERVIEW
the door to the discovery of the huge chemokine family, many of which have structural similarities
to platelet factor four8.
Today, Richmond continues to study MGSA and related chemokines. Her lab is currently
pursuing three different research projects:
MGSA transcription. Transcription is the first step in the process by which cells make proteins.
In this step the information coded in DNA is copied onto a single strand of RNA. The information
needed to form a given protein is cut out and consolidated into a strand of messenger RNA
(mRNA), which serves as the template for the actual assembly of the protein. Richmond is
investigating what triggers the transcription of MGSA mRNA and what shuts it off. If transcription
can be regulated, then the amount of MGSA that is produced can be controlled as well.
MGSA receptors. The researchers are characterizing receptors that recognize MGSA and thus
participate in the wound healing and tumor growth processes. If these receptors can be
understood in depth, then perhaps a way can be devised to keep them from reacting to excess
MGSA.
Wound healing and tumor growth. By investigating how normal cells heal, the researchers are
attempting to determine the natural mechanisms that start and stop the production of MGSA. By
using special chemokines9 that block the growth of new blood vessels to the tumor, the team is
testing ways to block tumors from growing.
Although much work remains before melanoma can be cured, Richmond’s outlook is positive. So
much advancement has been made in the field of chemokine research since the characterization
of MGSA, that new approaches to the problem are being designed every day. Each new assay 10
yields new information and a renewed chance to discover what makes tumors grow and how to
stop them. Even if the exploration of MGSA’s activities does not lead to a cure for melanoma, the
family of chemokines that it introduced could hold the answer to many of basic questions abut
how the body works at a molecular level.
8
Today approximately fifty chemokines have been characterized and many more are under investigation.
9
Angiogenic and angiostatic chemokines are small proteins that cause the production of new
blood vessels and those that counteract it. The two types of chemokines are very similar
molecularly: they differ by only a few amino acids. But their biological functions are directly
opposed to one another.
10
A scientific procedure that determines the quantity of a given substance present in a sample.
-3-
Ann Richmond's laboratory gains new insights into tumor growth and
wound healing through studies of the "SOS gene
BIOGRAPHY
A soda clerk turned chemistry teacher and a developmental biologist with the reputation as one of
the toughest and best members of his department are among the role models that have guided
Ann Richmond in her career as a successful cancer researcher.
As a schoolgirl growing up in a small town in Arkansas, Richmond relished challenging subjects.
For that reason, she found science interesting, but it didn’t occur to her that it could be a career.
There was one chemistry teacher, Mrs. Luke, who succeeded in fascinating Richmond in
quantum theory and other aspects of the world of atoms and molecules. Despite a longtime
interest in science, Mrs. Luke came to science teaching late in life. For many years, she was
content to work behind the counter of the local soda fountain. But then a change in her family
situation prompted her to get a teaching certificate and begin teaching chemistry at the local high
school.
Richmond recalls Mrs. Luke’s classes as the first science instruction that she truly enjoyed. The
experience convinced her to minor in science when she earned her teaching degree in college.
After she began teaching, Richmond realized that earning a master’s degree in chemistry would
be good for her career. So she signed up. While studying for her master’s, she found that she
really enjoyed research and so decided to get a doctorate.
“I liked science because it was challenging,” Richmond recalls. “I always liked the classes that
were the most difficult!”
Given her penchant for challenges, it wasn’t surprising that Richmond chose one of the toughest
mentors in the department of developmental biology at Emory University as her doctoral adviser.
Professor Bill Elmer had a reputation as one of the toughest and one of the best, she recalls.
Richmond remembers his interactive style in the lab as well as his effective teaching techniques.
“He was probably the most influential role model I had during my early scientific career.”
As she has continued to grow and mature as a scientist, however, Richmond has not limited her
role models to Elmer and Mrs. Luke. “I have developed a collage of role models, based on many
different men and women who have helped shape my scientific career, whom I have watched and
learned from.”
By the time Richmond was a postdoctoral fellow and beginning her work on MGSA, she was 33
years old with two small children. Lab life with its long hours and difficult problems was tough, but
Richmond thrived on the challenge. She describes a scientific career as something of a roller
coaster. At some points, the ride is fast and hard, but, at other points, the trip goes a little slower
so that one can focus her time and energy on other important things.
In the beginning years of the MGSA project, however, Richmond’s life seemed stuck on a fast
and hard stretch. The research and the research conditions were challenging. Richmond spent
many late nights in the library and the lab working on the MGSA puzzle. Part of her job was
interviewing melanoma patients, and those experiences constantly refueled her. Each interview
personalized the problem once again. Many of the patients were the same age as Richmond and
in similar situations. Many were young mothers whose lives seemed interchangeable with her
own.
Two patients in particular made a lasting impression. One was a young mother of two whose
illness progressed with startling rapidity. The woman was healthy enough to run the Peachtree
Road Race in Atlanta in July but died from melanoma in December. Richmond was particularly
touched by the woman’s story and the fate of her small children. The other was an older lady,
whose disease had spread to her lungs. Richmond spent an entire evening talking with her. “She
was warm and kind,” Richmond recalls, “The doctors said she had a bad case of the ‘oh-mylord’s,’ meaning that not only her body, but her will was tired of fighting the melanoma.” Richmond
left the patient that evening and when she returned to work the next morning she learned that the
woman had passed away during the night. Richmond’s job was then to extract tumor tissue
-4-
Ann Richmond's laboratory gains new insights into tumor growth and
wound healing through studies of the "SOS gene
BIOGRAPHY
during the autopsy. When she saw the woman’s body, she fainted. “It hit me that one day
someone will be there and the next day she won’t,” she recalls.
“These patients lit a flame,” Richmond explains, “one that would not go out and could not be
ignored.” Richmond relied on the stories of the people with whom she had talked for
encouragement when the research became difficult. Late at night, often with her girls asleep
beside her, memories of the men and women she had met who were fighting for their lives drove
her to finish just one more experiment on the borrowed equipment.
“I’ve never been a researcher without emotion,” Richmond says.
-5-
Ann Richmond's laboratory gains new insights into tumor growth and
wound healing through studies of the "SOS gene
BACKGROUND
DNA
DNA (deoxyribonucleic acid) is the master molecule of heredity. It is made up of two strands
linked together by four bases in the form of a double helix. Each strand of DNA is made up of a
backbone and bases that are strung in a precise order. The two strands of DNA come together
like a zipper. The teeth, or bases, on one side of the zipper match with the corresponding bases
on the other side. The bases are the nucleic acids adenine (A), guanine (G), cytosine (C), and
thiamine (T). Adenine and thiamine always pair together, as do guanine and cytosine. The four
bases are the letters in the alphabet of life.
Transcription
In the process called transcription, the two strands of DNA unzip so the bases are exposed. By
assembling bases in the same sequence as that of one of the DNA strands, the protein
machinery within the cell makes RNA, or ribonucleic acid. RNA is made up of the same four
bases as DNA (except thiamine, which is replaced by uracil (U)), but it is single stranded. So RNA
is a copy of half of the original DNA. Once the RNA is made, special proteins pare it down to the
information-carrying sections, which are called exons. The sections that are cut out are called
introns, or intervening sections. It is a process roughly analogous to editing video. Say a
vacationer with a video camera shot a lot of video footage and wants to edit it into a short
program to show friends and relatives. To do so, he must go through the footage and copy only
the best scenes onto a new tape. Similarly, the cell’s machinery extracts only the sequences it
needs to construct a given protein from the DNA and combines them in a temporary RNA copy,
which is called messenger RNA, or mRNA.
Translation
Once messenger RNA is produced, translation begins. Translation is the process that produces a
specific protein based on the blueprint contained in the mRNA. Proteins consist of chains of
amino acids. Each amino acid is coded for by a sequence of three bases called a codon. For
example, the codon containing the bases cytosine (A), guanine (G) and adenine (A) code for the
amino acid alanine. Complicating the picture is the fact that several codons specify the same
amino acid. Other codons mark the beginning and end of proteins: The codon AUG, for example,
is called the start codon because it signals the beginning of a protein sequence. When an mRNA
containing these instructions is released from the cell, it attaches to an organelle, called a
ribosome, that acts as a microscopic protein factory. The ribosome reads each codon and
attaches the correct amino acid to the growing protein molecule until it is completed.
Protein characterization
To identify the protein MGSA, Richmond had to reverse the normal translation and transcription
process. She began with the complicated process of purifying the protein from tumor cells. She
then used several different procedures that separated proteins by their charge and size to isolate
MGSA. Once she had enough purified MGSA, Richmond turned to a process called
microsequencing to determine the protein’s primary structure 11. Proteins are made of strings of
amino acids. To determine the order in which the amino acids are assembled, amino acids must
be knocked free from the end of the protein one by one and the identity of each amino acid
determined. When only a small amount of the protein is present, the amino acid sequence is
determined on a micro-scale, hence the term: microsequencing. This provides the order of the
amino acids. Since several codons code for each amino acid, however, Richmond was forced to
turn to yet another technology to determine the order of the bases in the messenger RNA: a
technology called molecular cloning from a cDNA library.
11
Proteins also have what are referred to as primary, secondary, tertiary and quaternary structures. The
primary structure refers to the order of the amino acids. Secondary and tertiary structures refer to how the
protein folds. Quaternary structure refers to the way that several proteins group together.
-6-
Ann Richmond's laboratory gains new insights into tumor growth and
wound healing through studies of the "SOS gene
BACKGROUND
cDNA Libraries
In order to determine the structure of proteins, researchers are interested in the messenger RNA
sequences that code for each amino acid. However, mRNA is unstable. It easily degrades under
normal temperatures and conditions, making it difficult to work with directly. So researchers
imitate nature by converting all the mRNA from a given source into DNA, which is extremely
stable. Because these are copies of DNA, researchers call them cDNA libraries. The scientists
use these libraries when they know portions of the amino acid sequence of a protein. From this
information, they make a few matching codon sequences and then use this as a “hook” to catch
the strands of DNA that contain matching sequences. Once they have retrieved the correct DNA
sequence from the thousands contained in a cDNA library, they can obtain the sequence from the
appropriate segment of the DNA and so nail down the gene’s exact sequence.
DNA extraction, digestion and PCR
Richmond’s team currently spends a portion of its time investigating the roles of different
chemokines in processes such as wound healing and tumor growth. To do so, the researchers
must know when the mice that they use as an animal model encode variant genes for specific
chemokines. They do this by extracting some DNA from the animal and testing it to see if it has a
given chemokine gene. First, a researcher removes a tiny bit of skin. Special chemicals are used
to digest everything but the DNA. The DNA is then run through a process called a polymerase
chain reaction. In the PCR process, the double-stranded DNA is heated just enough so that it
opens up and can be copied. Researchers can regulate which sections of the DNA will be copied.
By doing this repeatedly, a PCR machine can produce thousands of copies of a specific section
of DNA. The multiplied DNA is then mixed with a loading dye and put onto one end of a gel, a
material with the consistency of Jell-O that is spread about one-fourth-inch thick onto a sheet of
hard plastic. Electricity is run through the gel in order to exert pressure on the DNA molecules.
Smaller pieces of DNA move faster and so travel farther than the heavier ones. Once the gel has
run for several minutes a fluorescent chemical called ethidium bromide, or EtBr, is added. EtBr
mixes with the DNA and shows the DNA’s position when viewed under ultraviolet light. The test
allows researchers to identify specific fragments of DNA.
- VU -
-7-