Grantee Research Project Results
1999 Progress Report: "Patched" Gene Mosacism as a Basis for Differences in Skin Cancer Susceptibility
EPA Grant Number: R827018Title: "Patched" Gene Mosacism as a Basis for Differences in Skin Cancer Susceptibility
Investigators: Burns, Fredric J. , Sun, Tung-Tien , Shore, Roy E. , Roy, Nirmal
Institution: New York University
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 1998 through September 30, 2001 (Extended to September 30, 2002)
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $570,271
RFA: Interindividual Variation in Human Susceptibility to Environmentally-caused Disease (1998) RFA Text | Recipients Lists
Research Category: Human Health
Objective:
This research is intended to examine the role of the patched (PTCH) gene as a basis for differences in skin cancer susceptibility of people exposed to ionizing and/or ultraviolet radiation. Of about 2,000 people who were exposed to x-irradiation for tinea capitis at 8 years of age, about 10 percent have developed at least one skin cancer as of 35 years after exposure. Most interestingly, about 30 subjects have developed three or more skin cancers in comparison to the random expectation of only two such subjects. A high (90%) incidence of loss of heterozygosity (LOH) of the PTCH gene was found. Mutations in the PTCH gene lead to the autosomal dominant condition, known as nevoid basal cell carcinoma syndrome (Gorlin's syndrome). One way to understand the higher than expected occurrence of multiple basal cell carcinomas (BCCs) is to hypothesize that one PTCH allele was mutated in a skin progenitor cell during embryonic development. If so, the skin of an adult would be a genetic mosaic containing regions with a mutated PTCH allele. Multiple cancers then might be the result of the high sensitivity of such a mutated region to exogenous carcinogens, as occurs in Gorlin's syndrome. This hypothesis will be tested by examining normal skin epithelial cells in the immediate vicinity of PTCH-altered BCCs to determine if PTCH mutations are present; and if so, whether they are identical to mutations seen in the tumor itself.Progress Summary:
As of the end of the second year of this 3-year study, a vast majority of all samples have been collected from all three study sites, and analysis of all samples is well under way. Most of the RIAs to be performed by Oregon Graduate Institute and Oregon Regional Primate Center have been completed and most of the histology and immunocytochemistry have been completed. The organochlorine analysis is under way and all samples have been extracted and should be analyzed within the next 2 months. The samples for RIAs to be run at University of Florida have been delivered to them, but the samples await the completion of the development of the RIA for 1 -hydroxycorticosterone (which is behind schedule but should be completed within the next 2 months) before they will be run. The vertebra analysis (of age and growth) is completed on most samples. We fully expect to complete the entire project within the time frame proposed.Efforts to extract and purify vitellogenin (VTG) from bonnethead shark serum have been conducted and suggest low circulating levels of VTG in this species. This also has been demonstrated in another elasmobranch, the little skate, Raja erinacea (Perez and Callard, 1993). As a result, we have directed most of our effort toward the isolation and purification of the yolk protein lipovitellin, which Perez and Callard (1993) had used to produce antibodies for the detection of VTG in R. erinacea. Yolk proteins have been extracted following the methods described in Perez and Callard (1993), and we are in the process of validating the purification of putative bonnethead shark LPV via western blot. Once this is completed, antibodies will be produced against bonnethead LPV and used to detect the presence of VTG in serum. Treatment of male bonnethead sharks with 17 -estradiol has demonstrated the induction of a high molecular weight protein (putative VTG), which is not normally present in untreated animals. Although these results require validation, they strongly support the use of VTG as a marker for estrogenic compounds in this species.
At the current time, we have samples from 178 sharks from the Tampa Bay/Anclote River area, 155 from the Florida Bay area, and 92 sharks from the Apalachicola Bay area. Of the ones collected from Tampa Bay, they are divided as follows: 40 immature females, 46 mature females, 44 immature males, and 48 mature males. All sharks from this area have been collected. Of the sharks collected from Florida Bay, they are divided as follows: 41 immature females, 41 mature females, 32 immature males, and 41 mature males. All sharks from this area have been collected with the exception of 8 immature males. The immature males are the smallest individuals that we are seeking and they are often not captured by the net when all other groups are routinely captured. Of the sharks collected from Apalachicola Bay, they were divided as follows: 34 immature females, 12 mature females, 29 immature males, and 17 mature males. There are a number of mature animals that could not be collected from this area due to different migratory patterns. This means that mature animals are absent from the area during at least three of the eight stages to be collected.
The sample analysis phase of this study is well under way. We observed during our field collections, that infertility rates were much higher in the Tampa Bay/Anclote River area than in the Florida Bay area. We found 17 infertile ova that were distributed in 28 percent of the potential females captured in the Tampa Bay/Anclote River area and found only two infertile ova (one each in two females) in sharks from Florida Bay. We also observed that sperm viability from mated females was decreased in the Tampa Bay/Anclote River population as compared to the Florida Bay population. We further observed that sperm counts from males prior to mating and from females after mating and prior to ovulation from the Tampa Bay/Anclote River population were lower than those of the Florida Bay population. Regarding serum hormone comparisons, females, both mature and immature, from the Florida Bay population have a mean estradiol concentration that is double that of the females from the Tampa Bay/Anclote River population, although it is significant only in the immature females. Immature females from Florida Bay have a significantly higher testosterone concentration and immature males from Tampa Bay/Anclote River have a significantly higher dihydrotestosterone concentration. Serum samples from Apalachicola Bay have not been fully analyzed to include them in this comparison.
Analysis of age and growth have yielded some significant differences between the three populations. Sharks from the Florida Bay population are born smaller, grow more slowly, mature at a smaller size, reach a smaller maximum size, but the same maximum age as the Tampa Bay sharks. The sharks from the Apalachicola Bay population are born larger, grow more rapidly, mature at a larger size, reach a larger maximum size, but do not reach as high a maximum age as the Tampa Bay sharks. It is not known at this point whether these are latitudinal differences or are caused by something else.
As of the end of the second year of this 3-year study, a vast majority of all samples have been collected from all three study sites, and analysis of all samples is well under way. Most of the RIAs to be performed by Oregon Graduate Institute and Oregon Regional Primate Center have been completed and most of the histology and immunocytochemistry have been completed. The organochlorine analysis is under way and all samples have been extracted and should be analyzed within the next 2 months. The samples for RIAs to be run at University of Florida have been delivered to them, but the samples await the completion of the development of the RIA for 1 -hydroxycorticosterone (which is behind schedule but should be completed within the next 2 months) before they will be run. The vertebra analysis (of age and growth) is completed on most samples. We fully expect to complete the entire project within the time frame proposed.Efforts to extract and purify vitellogenin (VTG) from bonnethead shark serum have been conducted and suggest low circulating levels of VTG in this species. This also has been demonstrated in another elasmobranch, the little skate, Raja erinacea (Perez and Callard, 1993). As a result, we have directed most of our effort toward the isolation and purification of the yolk protein lipovitellin, which Perez and Callard (1993) had used to produce antibodies for the detection of VTG in R. erinacea. Yolk proteins have been extracted following the methods described in Perez and Callard (1993), and we are in the process of validating the purification of putative bonnethead shark LPV via western blot. Once this is completed, antibodies will be produced against bonnethead LPV and used to detect the presence of VTG in serum. Treatment of male bonnethead sharks with 17 -estradiol has demonstrated the induction of a high molecular weight protein (putative VTG), which is not normally present in untreated animals. Although these results require validation, they strongly support the use of VTG as a marker for estrogenic compounds in this species.
At the current time, we have samples from 178 sharks from the Tampa Bay/Anclote River area, 155 from the Florida Bay area, and 92 sharks from the Apalachicola Bay area. Of the ones collected from Tampa Bay, they are divided as follows: 40 immature females, 46 mature females, 44 immature males, and 48 mature males. All sharks from this area have been collected. Of the sharks collected from Florida Bay, they are divided as follows: 41 immature females, 41 mature females, 32 immature males, and 41 mature males. All sharks from this area have been collected with the exception of 8 immature males. The immature males are the smallest individuals that we are seeking and they are often not captured by the net when all other groups are routinely captured. Of the sharks collected from Apalachicola Bay, they were divided as follows: 34 immature females, 12 mature females, 29 immature males, and 17 mature males. There are a number of mature animals that could not be collected from this area due to different migratory patterns. This means that mature animals are absent from the area during at least three of the eight stages to be collected.
The sample analysis phase of this study is well under way. We observed during our field collections, that infertility rates were much higher in the Tampa Bay/Anclote River area than in the Florida Bay area. We found 17 infertile ova that were distributed in 28 percent of the potential females captured in the Tampa Bay/Anclote River area and found only two infertile ova (one each in two females) in sharks from Florida Bay. We also observed that sperm viability from mated females was decreased in the Tampa Bay/Anclote River population as compared to the Florida Bay population. We further observed that sperm counts from males prior to mating and from females after mating and prior to ovulation from the Tampa Bay/Anclote River population were lower than those of the Florida Bay population. Regarding serum hormone comparisons, females, both mature and immature, from the Florida Bay population have a mean estradiol concentration that is double that of the females from the Tampa Bay/Anclote River population, although it is significant only in the immature females. Immature females from Florida Bay have a significantly higher testosterone concentration and immature males from Tampa Bay/Anclote River have a significantly higher dihydrotestosterone concentration. Serum samples from Apalachicola Bay have not been fully analyzed to include them in this comparison.
Analysis of age and growth have yielded some significant differences between the three populations. Sharks from the Florida Bay population are born smaller, grow more slowly, mature at a smaller size, reach a smaller maximum size, but the same maximum age as the Tampa Bay sharks. The sharks from the Apalachicola Bay population are born larger, grow more rapidly, mature at a larger size, reach a larger maximum size, but do not reach as high a maximum age as the Tampa Bay sharks. It is not known at this point whether these are latitudinal differences or are caused by something else.
As an initial group designed to establish proof-of-principle, 14 subjects were selected as likely to have either single or multiple BCCs. Of these patients, seven expected to have multiple cancers and seven expected to have single cancers. All 14 were brought into the clinic and examined for the presence of BCCs and other skin lesions in the head and neck regions. Three subjects exhibited no skin cancers, although two of these had existing lesions removed in the preceding year during visits to their own dermatologist, and one patient refused to provide a blood sample, leaving a total of 10 informative subjects (i.e., subjects for whom both cancer and blood DNA are available). Out of these 10 subjects, a total of 19 BCCs were found and biopsied. A few individuals had their tumors biopsied at NYU and then refused further treatment at the NYU Clinic. For one such subject, cancer and normal skin tissue was obtained from an excision performed by the subject's private physician. Surprisingly, none of the subjects elected to have the MOHS procedure, which involves microscopic removal of progressive slices of tissue until no cancer cells can be detected, apparently due to the complexity of the procedure in comparison to a simple excision.
DNA has been extracted from 19 adequate part one tumor DNA samples and the corresponding normal bloods. LOH analysis has been performed on this DNA for six microsatellite markers in the region of chromosome 9q22.3. The DNA analyses have not yet been completed on the normal skin samples, largely because of the small amount of normal tissue (and DNA) obtained in the dissection procedure. Initial results show rearrangements near the patched locus in 12 of 19 BCCs (63%). This percentage is a little lower than the 9 of 10 (90%) found in our initial survey, but the difference is not statistically significant; however, the location of rearrangements varied somewhat from that seen in the initial survey (i.e., there appears to be a greater incidence at specific markers than seen in the previous study).
A possibly related finding to this second goal is the existence in 2 of 10 patients of a third microsatellite band at D9S 280 in blood DNA, suggesting that these people may be mosaics at this chromosome location. If true, this mosaicism must have been established early in embryonic development, because it affects approximately 50 percent of adult blood cells. One individual has two BCCs that show LOH patterns differing from those seen in the blood, indicating that in the BCCs the allele pattern has reverted to two alleles. These are extremely preliminary findings, but suggest that this mosaicism may be present and detectable in the blood as well as in the skin. Further studies are being conducted to confirm and extend these very interesting early findings relating possible mosaicism in blood to similar patterns in the BCCs.
Future Activities:
A second major goal is to establish the DNA sequence changes that inactivate the PTCH gene in people developing skin cancers as a result of exposure to ionizing radiation. Cancer tissue has been separated from the normal skin microscopically and is undergoing analysis to determine the fraction of each sample that is "normal" based on histological examinations. If the hypothesis that a mosaic of mutated cells surrounds or underlies the cancers is correct, then these mutations must have been the first ones in the sequence that produced the tumor. It is hoped that these data will help to identify the precursor cell population for BCCs as stem cells in a bulb in the central region of each hair follicle matrix or dedifferentiated squamous cells in the epidermis.
The anticipated model enhancement activities include: testing and assessment of alternate aerosol equilibrium modules; interfacing the multiscale cloud-chemistry package with the MAQSIP gas-aerosol model; investigating potential improvements in dry-deposition velocity estimates for NH3; and initiating the development of an inverse modeling methodology.
Model simulations with the updated emissions will be conducted to: (1) study the sensitivity to grid resolution; (2) determine range of influence of reduced and oxidized nitrogen; and (3) study impact of potential reductions in NOx and increases in NH3 emissions on PM composition.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other project views: | All 4 publications | 3 publications in selected types | All 1 journal articles |
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Type | Citation | ||
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Zhao P, Zhu X, Liu Y, Wang B, Wang C, Burns FJ. Solar ultraviolet radiation and skin damage: An epidemiological study among a Chinese population. Archives of Environmental Health 1998;53:405-409. |
R827018 (1999) R827018 (2000) |
not available |
Supplemental Keywords:
ultraviolet radiation, risk assessment, sensitive populations, carcinogen, children, genetic predisposition, susceptibility., RFA, Health, PHYSICAL ASPECTS, ENVIRONMENTAL MANAGEMENT, Ecosystem Protection/Environmental Exposure & Risk, Ecosystem/Assessment/Indicators, Ecosystem Protection, Risk Assessments, Disease, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Physical Processes, Ecological Indicators, Risk Assessment, health effects, deterministic linkages, ecological effects, ecological risk assessment, biomarkers, assessment models, gene-environment interaction, environmental stressor, exposure, patched gene mosaic, adverse human health affects, environmental consequences, environmental mutagens, patched gene, human exposure, susceptibility, ecosystem indicators, environmental stressors, assessment methods, biological markers, skin cancer, biomedical research, exposure assessment, cancer risk, skin cancer susceptibilityProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.