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Molecular Oncology, Markers, Clinical Correlates |
Virology Division, Department of Microbiology, South Eastern Sydney Area Laboratory Services, The Prince of Wales Hospital, Randwick, NSW 2031, and School of Biotechnology and Biomolecular Science, University of New South Wales, Kensington, Australia [C. E. F., Y. D., W. D. R.]; Immunohistochemistry Division, Department of Anatomical Pathology, St. Vincents Hospital, Darlinghurst, Australia [D. T.]; Department of Pathology, National Cancer Hospital, Hanoi, Vietnam [V. T. T.]; and School of Public Health, University of New South Wales, Kensington, Australia [J. S. L.]
| ABSTRACT |
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Experimental Design: Using PCR and primers that amplify MMTV-like gene sequences, we tested cancerous and benign breast tissue from Caucasian-Australian, Vietnamese-Australian, and Vietnamese women.
Results: MMTV-like gene sequences were amplified in 19 of 45 (42.2%) archival breast cancer biopsy tissues from Caucasian-Australian women, but only 1 of 120 (0.8%) and 0 of 41 breast cancer biopsy tissues from Vietnamese and Vietnamese-Australian women, respectively. The same sequences were found in only 2 of 111 (1.8%) and 0 of 60 normal (benign) breast tissue samples from Australian and Vietnamese women, respectively.
Conclusions: MMTV-like gene sequences are found in only some human populations and are rarely found in normal human breast tissue from all populations, suggesting they are not present in the normal human genome and have been acquired.
| INTRODUCTION |
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| MATERIALS AND METHODS |
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Breast tumors were diagnosed according to standard criteria (9) . Cancerous epithelial breast cells were identified using H&E stains (10) . Human breast tissues were processed in a class II laminar flow hood, located in a separate laboratory to where PCR was performed. Paraffin was removed from tissues with two washes of xylene, supernatant removed, and the pellet washed in 100% ethanol. Tissues were resuspended in 100 µl of digestion buffer (150 mM NaCl, 15 mM Tris-HCl, 1 mM EDTA, and 0.1% SDS) with 5 µl of proteinase K (20 mg/ml), incubated at 55°C for 3 h, and then at 95°C for 10 min to inactivate the proteinase K. Phenol-chloroform extraction was performed, the DNA precipitated in ethanol, and the supernatant removed. The pellet was dried and resuspended in 20 µl of 10 mM Tris (pH 8)-1 mM EDTA buffer containing RNase, and the DNA concentration determined using a spectrophotometer.
Nested PCR was performed using a Perkin-Elmer 9700 thermal cycler and Taq polymerase (Promega) with minor modifications to published techniques, and primers using standard precautions and controls (3 , 4 , 11) . Positive controls consisted of DNA extracted from the MCF-7 human breast cancer cell line that expresses MMTV env gene-like sequences (4) , and DNA quality was assessed by amplifying extracted DNA with p53 and ß-globin primers (12 , 13) . Cycle conditions for MMTV outer PCR with primers 5L and 3L (4) were 94°C for 2 min, 30 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 40 s, followed by 72°C for 3 min. Ten µl of outer product was then added to the inner reaction mix using primers 1X and 2NR (4) . PCR was performed at 94°C for 2 min, 30 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 30 s, followed by 72°C for 3 min. PCR products were analyzed using agarose gel electrophoresis, and all of the positive PCR results for MMTV-like gene sequences were repeated. PCR products were sequenced and sequences analyzed as described previously (14) .
Power calculations on sample size were performed using standard calculations (15) , and the association between MMTV-like gene sequences and breast cancer assessed using Fishers exact test.
| RESULTS |
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| DISCUSSION |
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A significant difference in the prevalence of antibodies reactive with MMTV and breast cancer rates has been reported between women from Western and Eastern countries (17) , with the prevalence of antibodies reactive with MMTV in the sera of women with breast cancer found to vary from <5% in mainland Chinese, 18.6% in North Americans, 38% in Indians, and 61.9% in East African women (17) . Antibody levels reported in Chinese women are similar to the extremely low prevalence levels of MMTV-like gene sequences in Vietnamese women as reported in this study (<1%).
It is interesting that the breast cancer tissues from the first generation Vietnamese-Australian women did not contain MMTV-like gene sequences. This is similar to the low levels detected in Vietnamese women from Hanoi, and unlike the high levels reported in Caucasian-Australian women. It would be interesting to examine the prevalence of MMTV-like gene sequences in the next generation of Vietnamese women born in Australia, to additionally investigate the impact of geography and environment on MMTV-like gene sequences and breast cancer.
The differences in breast cancer rates and prevalence of MMTV-like gene sequences between populations may be related to host factors and the geographic distribution of different mouse species (6) , as exogenous MMTV is found in up to 50% of Mus domesticus from Western countries (18) . It has been hypothesized that MMTV may have the ability to infect across species (6 , 18) . The high prevalence of MMTV-like gene sequences in Australian women (similar to United States women) and low prevalence in Vietnamese women (both in Australia and Vietnam) may support this hypothesis.
Histopathological analysis of the Australian breast tissues screened showed an increasing prevalence of MMTV-like gene sequences with severity of cancer. This suggests that these MMTV-like gene sequences are associated with more invasive breast carcinoma and may be involved in the etiology of human breast cancer.
There is recent evidence that suggests that these MMTV-like gene sequences may be of exogenous origin (19 , 20) . The finding of MMTV-like gene sequences in breast cancer tissue, but rarely in normal breast tissue, supports this hypothesis.
Our findings confirm that MMTV-like sequences occur in a high proportion of women with breast cancer in some human populations and are rarely found in normal breast tissue. This supports the contention that these sequences represent a virus associated with human breast carcinogenesis and not a normal part of the human genome.
| ACKNOWLEDGMENTS |
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| FOOTNOTES |
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1 Supported in part by the Australian Cancer Council and the Medical Benefits Fund of Australia. ![]()
2 To whom requests for reprints should be addressed, at Virology Division, Department of Microbiology, South Eastern Area Laboratory Services, The Prince of Wales Hospital, Randwick NSW 2031, Australia. Phone: 61-2-93829050; Fax: 61-2-93984275; E-mail: w.rawlinson{at}unsw.edu.au ![]()
3 The abbreviations used are: MMTV, mouse mammary tumor virus. ![]()
Received 5/14/02; revised 11/ 4/02; accepted 11/ 6/02.
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