Editor's note: This "Under the Scope" is the first in a series of profiles on areas of intramural research at the NIEHS.
The secrets to the origins of cancer are hidden deep within cells. Researchers in the Laboratory of Experimental Pathology (LEP) at the NIEHS are using the techniques of molecular biology to uncover the role of environmental agents in changing the molecular structure of cells that may lead to cancer.
Traditional pathology consisted of using stained tissue sections for diagnosis and for identifying specific tissue alterations such as identification of infectious organisms and demonstration of specific cellular enzymes. A second generation of specialized stains based on immunohistochemistry provided information about the presence and localization of specific proteins for which polyclonal and monoclonal antibodies were available. Today, molecular pathology laboratories are using molecular methods such as
in situ
hybridization staining to detect specific messenger RNAs. These methods allow a researcher to show whether a cell has actually produced a protein, rather than just stored it. Because changes in gene expression (indicated by changes in mRNA) are associated with induction and progression of tumors,
in situ
hybridization offers scientists a means of teasing out the underpinnings of the cancer process. Other dimensions of molecular pathology including extraction of cellular DNA and RNA as well as proteins from tumors and identification of this material using electrophoresis and other molecular biology techniques have led to identification of several oncogenes in human and animal cancers.
Oncogene Clues
Lung cancer is one of the most common cancers in the United States. Worldwide, liver cancers are the most common. Most animal carcinogenicity studies have been conducted in mouse lung or liver cells because mice readily develop tumors in these sites when exposed to carcinogenic agents. Efforts led by Robert Maronpot, chief of the LEP, are ongoing in the laboratory to better understand these tumor endpoints and their utility in hazard identification and human risk assessments. "We hope that there are bridges between tissues and across species that will enable us to transfer this information to humans," Maronpot said. "If something is occurring molecularly, then we might find a way to block it, thus blocking cancer, or provide methods for better therapies."
LEP researchers. (left to right) Akiko Enomoto, Kathy Phillips, Dave Malarkey, Robert Maronpot, and Barbara Davis. (Not pictured is Darlene Dixon.)
Early molecular pathology studies conducted by the LEP in collaboration with other NIEHS researchers looked at DNA from tumor cells and adjacent tissues in mice for alterations to proto-oncogenes, normal genes that mutate slightly to become oncogenes and cause cancer. After identifying a gene, the investigators treated the animals with an environmental agent to determine if the gene's activity changed, such as causing more tumors to develop than in normal mice or causing tumors to develop sooner. For example, researchers found that tetrachloroethylene and trichloroethylene, widely used industrial solvents and common contaminants of surface, ground, and drinking water, and dichloroacetic acid, a metabolite of trichloroethylene and a major organic contaminant of chlorinated drinking water, activated oncogenes in induced liver tumors. This activation led to an early increase in liver tumor incidence in one strain of mice. Some of the activated oncogenes in these tumors differed from those found in tumors that occurred spontaneously.
Past studies have shown that oncogene activation is more frequent in tumors induced by low doses of hepatocarcinogens than by high doses and provides evidence that the mechanisms or pathways of tumor induction differ as a function of the dose of carcinogen. Dave Malarkey is currently studying the dose-response relationship to oncogene activation. The implications of identifying a dose-dependent effect on the mechanism of tumor induction directly challenge the risk assessment assumption that chemical carcinogens at low and high doses produce cancer by the same mechanisms.
Most of the oncogene studies in mouse liver tumors have been done at NIEHS, says Maronpot. However, the focus is shifting away from these studies because not all induced or spontaneous liver tumors have been shown to carry an activated oncogene, making it difficult to draw definitive conclusions. Some of these types of studies will continue though, in the hopes of discovering new oncogenes. "We determined that some agents that cause cancer in animals, and presumably in man, do so by activating oncogenes, which may tell us something about risk assessment. Also, some of the specific mutations in activated oncogenes identified in animal tumors match what is seen in human tumors, which offers the hope that the basic fundamental molecular mechanisms may be similar," said Maronpot.
In addition, retrospective studies are being designed to identify specific genetic alterations in neoplasms from some of the 450 previous two-year chemical bioassays. These studies will attempt to correlate chemical-specific properties including structural features, genotoxicity, and metabolism with characteristics of genetic alterations in preneoplastic and neoplastic lesions of specific target organs. These studies will allow scientists to compare classes of chemicals, evaluate structure-activity relationships within classes, and determine the response of target tissues to different chemicals without having to do long-term studies. Chemicals for which genetic alterations (oncogene activation) have been determined include oxazepam, vinyl carbamate, chlordane, trichloroethylene, tetrachloroethylene, dichloracetic acid, furan, and butadiene.
The Transgenic Track
A new focus is on transgenic mice. The mice are genetically engineered to carry human or mouse oncogenes or genes for certain growth factors. "They offer a unique approach to identifying and investigating factors that may influence tumor development by allowing us to directly assess what these genes do in target organs and how they are influenced by environmental agents," Maronpot says. Some transgenic mice develop tumors in as little as 6 months when exposed to certain chemicals, as opposed to the normal 18- to 24-month time span. The mice hyperrespond to test agents, enabling the investigators to obtain answers to their questions much faster than with traditional models.
LEP investigators will soon begin studying a strain of transgenic mice developed in Japan that carries a normal human H-ras
proto-oncogene. When these mice were exposed to potent carcinogens, this gene was activated in the induced tumors. Maronpot and his colleagues will expose the mice to environmental chemicals to which people may be exposed such as chloroform, chlordane, ethyl acrylate, and furan, which have previously been shown to cause cancer in laboratory animals. If the mice respond as expected, they will be used to test agents whose effects are unknown. "The questions are, however, Are we going to miss something with this technique or will we be overpredicting which agents cause cancer?" Maronpot says. "So far, these mice show a lot of promise. If we show that environmental agents produce tumors containing an activated human H-ras
gene, then we could warn people about their exposure."
Sleuthing
in situ
A relatively new area of investigation involves
in situ
hybridization, which allows researchers to identify proteins produced by specific genes in the tissue where these proteins are normally found.
Most of the current LEPin situ
studies involve mouse ovarian toxicity and cancer. Barbara Davis, a guest researcher in the LEP, and researcher Kathy Phillips are studying estradiol, a hormone neccesary for reproduction, in ovarian granulosa cells, which line the egg-containing follicles, to determine the effect of environmental agents on estradiol production. Because estradiol production is the same in animals and humans, it may be a good system for comparing toxicity.
Specifically, Davis is investigating how environmental agents act on aromatase, an enzyme that converts testosterone to estradiol and also acts on the estrogen and androgen receptors. The studies are performed in whole tissue and then
in situ
in specific rat ovarian cells. The primary agents under investigation are di(2-ethylhexyl)phthalate, which rat studies have shown suppresses estradiol production, and other phthalates to see if this class of agents has the same actions. "Phthalates are ubiquitous in the environment," Davis says. Phthalates are components of plastics and readily leech into the environment.
Ovarian variation. Using in situ hybridization, researchers can study expression of the oncogeneWT1 in ovarian cells.
LEP and other NIEHS researchers will also study mercury vapor using
in vivo
rat studies and
in vitro
rat and human granulosa cells. "We will look at these cells
in vitro," Davis said. "If we determine that mercury disrupts the pathways, then it implies that these pathways are likely to be disrupted in humans also." A 1994 study by NIEHS epidemiologist Andrew Roland found that female dental assistants with high occupational exposure to mercury (by making mercury amalgam tooth fillings) were less fertile than unexposed dental assistants.
Davis is using
in situ
hybridization studies to generate markers to identify the genes affected in ovarian cancer. There are not many studies on agents that cause ovarian cancer, although ovarian cancer is the fifth leading cause of cancer death in women. "If we better understood the underlying mechanism that causes ovarian cancer, we could develop preventative measures," Davis said. Davis and Phillips are studying theWT1
marker, an oncogene expressed in immature and adult ovarian cells, using an
in situ
method developed by pathologist Akiko Enomoto. They are also investigating the tumor-suppressor gene p53, and
BRCA1, an oncogene involved in inherited breast tumors as well as in ovarian cancer. These studies are being conducted using molecular probes, synthetic complementary copies of DNA sequences used to look for messenger protein expression.
These studies use
in situ
polymerase chain reaction (PCR), which allows the researchers to make millions of copies of any DNA sequence.
Signs of Promise
Other ongoing research areas in the LEP include the development of immunohistochemistry methods for cell products such as p53, estrogen receptors, fibroblast growth factors (FGF-ß), and other products related to cell growth and proliferation. The reproductive pathobiology group of the LEP, led by Darlene Dixon, is using immunohistochemical staining of mouse and human tissues to assess the presence of several growth factors in uterine tumors. Preliminary findings show that one particular growth factor, TGF-
is found in uterine smooth muscle tumors and expression increases with malignancy. Immunolocalization of a mature form of this growth factor has been found exclusively in malignant uterine smooth muscle tumors in mice.
Other techniques are under development such as
in situ
polymerase chain reaction (PCR) which will allow the amplification of specific sequences of DNA and RNA present in small amounts in individual cancerous cells. The technique enables researchers to amplify or make copies of a gene inside a cell, without destroying it, attach a color marker, and identify where a "bad" gene is located. "We want to understand how genes are activated, and when that occurs in the process in intact tissue, not tissue extract." Maronpot says. "This technique will allow us to identify damaged cells. Although many cells are exposed to the environmental agent, only some change, others die or are repaired. But the cell that did not die or repair itself, perpetuates the damage." Maronpot stresses that the contributions of molecular pathology must be combined with those of scientists in many disciplines including chemistry, toxicology, and physiology to unlock the secrets of how environmental agents affect human cells.
Barbara Proujan
Barbara Proujan is a freelance journalist in Graham, North Carolina.
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Last Update: July 16, 1997