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SV40 Detection in Human Tumor Specimens

Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, Illinois

Maria Ramos Niño and Brooke Mossman

Department of Pathology, University of Vermont, Burlington, Vermont

Harvey I. Pass

Thoracic Oncology, Karmanos Cancer Center, Detroit, Michigan

To the Editor:

Cristaudo et al. (1) provided the first molecular epidemiologic evidence supporting cocarcinogenesis between asbestos and SV40 in human malignant mesothelioma. The authors have also proposed that an inadequate technical procedure to detect SV40 could produce false-positive or false-negative results. Here we provide information for the optimization of the experimental procedure and to prevent laboratory artifacts. It is hoped that this information will help those interested in testing human specimens for SV40.

When testing tumor samples by PCR, it is important to rule out PCR and plasmid contamination. The precautions we recommend to prevent and to detect PCR contamination were described previously (2, 3). To test for laboratory contamination by plasmids, we recommend to run PCR reactions using the primers 5'-GCTCACGCTGTAGGTATCTC-3' and 5'-TCTAGTGTAGCCGTAGTTAG-3' that amplify a 241-bp portion of the pUC origin of replication present in pBR313 (4) and in virtually all plasmids that are propagated in E. coli (BLAST analysis; http://www.ncbi.nlm.nih.gov/BLAST/).

In Fig. 1A we show how the sensitivity of the detection method can drastically change the scoring of SV40 large T antigen (Tag) expressing malignant mesotheliomas (from 0% to 50%) in immunoprecipitation/Western blots. Note that A1 to A3 contain the same tumor lysates, and that the same membrane (A2) was stripped and rehybridized (A3). This experiment shows how apparently conflicting results can be resolved by optimizing the technical approach (see figure legend).

View larger version (69K): [in this window] [in a new window] [Download PPT slide]   Figure 1. A, immunoprecipitation/Western blot for Tag expression in 20 malignant mesothelioma specimens. Five hundred micrograms of total tumor lysate were immunoprecipitated with agarose-conjugated Pab 101 (Santa Cruz Biotechnology, Santa Cruz, CA). Precipitates were analyzed by Western blot following standard procedures, using Pab 419 as the primary antibody. F1, positive control, SV40-transformed human mesothelial cell line. To avoid that the positive control would capture most or all of the anti-Tag (and cause false-negative results), the strip of membrane containing F1 was cut out and hybridized separately and in parallel with those containing tumor samples. A1, an anti-mouse goat polyclonal serum conjugated to horseradish peroxidase (HRP) was used as the secondary antibody. A2, an anti-mouse, biotinylated chicken polyclonal antibody was used as the secondary antibody. The filters were then incubated with the avidin-biotin complex (Vectastain) reagent (which consists of a multimeric complex of streptavidin and biotinylated HRP). A3, the membranes in A2 were stripped of antibodies. After blocking and incubation with the primary antibody, an anti-mouse biotinylated chicken polyclonal antibody was used as the secondary antibody. The filters were then incubated with HRP-conjugated streptavidin. Bands were visualized by enhanced chemiluminescence. Note that using the procedure in A1, all specimens were negative because the method is not sufficiently sensitive. Using the ultrasensitive ABC reagent (A2), the low signal-to-noise ratio (i.e., after 5 seconds of exposure, the membrane is completely black, preventing testing different exposure times) allows the identification of only two positive samples: 65 and 76 (weakly positive). By using the intermediate sensitivity method in A3 (which allows to test different exposure times), it was possible to identify as Tag positive samples 55, 56, 58, 60, 65, 76, and 77. The signal was too low in 51 and 67 to make a definitive conclusion. Sample 71 showed a clear band of slightly higher molecular weight. This band may represent a mutated or differentially modified Tag. B, immunohistochemistry reactions done in parallel. Left, SV40 Tag nuclear staining (Pab 101, Santa Cruz) followed by polyclonal alkaline phosphatase–conjugated anti-mouse immunoglobulin G2A (Southern Biotech, Birmingham, AL). After the reaction was developed (with Fast Blue BB, Sigma, St. Louis, MO), the specimens were incubated with CD44 [DF1485 (Zymed, San Francisco, CA), followed by anti-mouse immunoglobulin G1A (Southern Biotech)], and stained with the 3-amino-9-ethylcarbazole reagent (Sigma; membrane staining, pale red). Right, immunohistochemistry of a consecutive slide of the same specimen shown in left. In this experiment, the two primary antibodies were mixed together, then the slide was incubated with the two secondary antibodies. Color development was done as described in left.

The described issues are in addition to other variables known to affect SV40 detection results, such as DNA extraction protocols and the relative content of cancer cells in a tumor specimen (reviewed in ref. 5). Our data support Cristaudo et al.'s message that only when rigorous controls are used is it possible to reliably evaluate the presence and expression of SV40 in human specimens. The examples shown underscore how small technical details can produce opposite results that at first glance could erroneously appear in conflict.

References

1. Cristaudo A, Foddis R, Vivaldi A, et al. SV40 enhances the risk of malignant mesothelioma among people exposed to asbestos: a molecular epidemiologic case-control study. Cancer Res 2005;65:3049–52.[Abstract/Free Full Text]
2. Carbone M, Pass HI, Rizzo P, et al. Simian virus 40-like DNA sequences in human pleural mesotheliomas. Oncogene 1994;9:1781–90.[Medline]
3. Rizzo P, Di Resta A, Powers A, et al. The detection of simian virus 40 in human tumors by polymerase chain reaction. Monaldi Arch Chest Dis 1998;53:202–10.[Medline]
4. Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 1977;2:95–113.[Medline]
5. Carbone M, Bocchetta M, Cristaudo A, et al. SV40 and human brain tumors. Int J Cancer 2003;106:140–2.[Medline]

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				B. Kroczynska, R. Cutrone, M. Bocchetta, H. Yang, A. G. Elmishad, P. Vacek, M. Ramos-Nino, B. T. Mossman, H. I. Pass, and M. Carbone Crocidolite asbestos and SV40 are cocarcinogens in human mesothelial cells and in causing mesothelioma in hamsters PNAS, September 19, 2006; 103(38): 14128 - 14133. [Abstract] [Full Text] [PDF]

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