Evaluation of Neuroblastoma Cell Lines for the Presence of SV40 Tag DNA




Deanne Cevasco

Introduction

Simian virus 40 (SV40), a polyomavirus of rhesus macaque origin, was discovered in 1960 as a contaminant of polio vaccines that were distributed to millions of people from 1955 through early 1963 (1). SV40 is a potent DNA tumor virus that induces tumors in rodents and transforms many types of cells in culture, including those of human origin (1). SV40 has been detected in human brain tumors such as ependymoma and choroid plexus tumors (2,3,4) as well as human papillary thyroid carcinomas and osteosarcomas (5,6). In addition, children who would not have been exposed to the contaminated vaccines, have developed tumors positive for SV40-like DNA sequences (3). This discovery has led to several hypotheses regarding the source of SV40 infection in humans. It has yet to be shown that the virus can be transmitted between humans, so the question of whether or not SV40 infection in humans was present prior to the introduction of the contaminated vaccines still remains.
The viral replication protein, named large T antigen (Tag), is also the viral oncoprotein (1). It is a nuclear phosphoprotein containing 708 amino acids (6). It disrupts cell growth control mechanisms, primarily by binding to and abolishing the normal functions of tumor suppressor proteins p53 and pRB family members (1, 7, 8, 9).
Because of the possible public health risk SV40 infection in humans poses, it is important to study the virus in order to determine the origin of its DNA sequences found in humans. The better the screening techniques, the more information will become available to find the cause of SV40 DNA integration found in human tumors.
In this study, four neuroblastoma cell lines and COS-7 cells, used as a positive control, were evaluated by PCR for the presence of SV40 Tag sequences. The PCR products from COS-7 cells were analyzed by DNA sequence analysis to confirm the presence of SV40 Tag DNA sequences.

Figures


Figure 1-PCR Products from Four Neuroblastoma Cell Lines and COS-7 Cells. Lane 2 shows the PCR product generated from COS-7 cells and primers PYV.for and PYV.rev as described in Materials and Methods. Lanes 3, 4, 5, 6 and 7 show no PCR products in BE(2)-C, KCN-69n, LA1-55n and SH-SY5Y cell lines and the negative control, respectively, with primers PYV.for and PYV.rev. Lanes 1, 8, 9, 10 and 11 are the PCR products generated from COS-7, BE(2)-C, KCN-69n, LA1-55n and SH-SY5Y cell lines with actin primers.


Figure 2-Lane 1 is the second amplification of the PCR product found in COS-7 cells in A. Lanes 2, 3, 4, 5 and 6 show the PCR product generated in COS-7 cells and the negative results in BE(2)-C, KCN-69n, LA1-55n and SH-SY5Y cell lines, respectively, using primers TA1 and TA2 as described in Materials and Methods.


Figure 3-DNA Sequence of the PCR Products from COS-7 Cells. The sequences read were reversed and complemented to align with the published SV40 complete genome sequence (GENBANK/J02410). (A) Sequence read from the PCR product generated from primers TA1 and TA2. (B) Sequence read from the PCR product generated from primers PYV.for and PYV.rev. The five identifiable nucleotide differences are a result of the differences in the primer sequences to the published sequence.


RESULTS

Detection of SV40 Tag Coding Sequences in COS-7 Cells. Primers PYV.for and PYV.rev (3, 4) were used to amplify a 172-bp sequence of SV40 Tag as described in materials and methods. A PCR product was detected in COS-7 cells but not in any of the four neuroblastoma cell lines (Fig.1A). Actin primers were also used on all the DNA samples to show that all of the DNA samples could be amplified by PCR. Because of the small amount of product detected in the COS-7 cells, the amplified DNA was used as the template for a second PCR reaction. However, it does not appear that the DNA was amplified further (Fig. 1B). In addition, a second set of primers, TA1 and TA2 (2), were used to amplify a 309-bp sequence of Tag. The four neuroblastoma DNA samples and COS-7 DNA were again evaluated for the presence of SV40 as described (Fig. 1B). SV40 Tag DNA was detected in the COS-7 cells only, which is consistent with the results of the first PCR experiment.
DNA Sequence Analysis of the Amplified PCR Products. The two PCR products amplified from COS-7 cells were analyzed by DNA sequencing (Fig. 2). The sequence amplified by primers PYV.for and PYV.rev, was homologous to the SV40 Tag sequence corresponding to nucleotides 4407 to 4572 of the complete virus genome (Fig. 3B). The differences in nucleotides 4410, 4413, 4554, 4569, and 4571 are due to inconsistencies in the primers to the published SV40 DNA sequence. Nucleotide 4557 was unable to be identifed due to the resolution of the sequencing gel.
The 309-bp sequence amplified by primers TA1 and TA2 was not sequenced in its entirety. Two partial sequences, 113-bp and 114-bp long, were homologous to nucleotides 2688 to 2801 and 2901 to 3015 of the SV40 genome, respectively (Fig. 3A). Seven nucleotides in total were unable to be identified in the two partial sequences due to the resolution of the sequencing gel.

DISCUSSION

Although SV40 Tag DNA was not found in the four neuroblastoma cell lines, the amplification and sequencing of PCR products from the COS-7 cells shows that the procedures used were successful in evaluating different DNA samples for SV40 DNA sequences. The negative results in the neuroblastoma cell lines were not due to inability of the DNA samples to be amplified by PCR or improper binding of the primers used.Both of these factors were ruled out by the use of actin primers in all samples (Fig. 1A) and the PCR products produced in the COS-7 cells which were shown to be homologous to SV40 Tag sequences (Fig. 3). Primers PYV.for and PYV.rev have been used in previous studies (3, 4) and the differences in the primer sequence and the published sequence is most likely due to the sequence differences known to occur between different strains of the virus (1, 2, 4). In two recent studies, variation in the C-terminal end of the T-antigen genes was found, and it has not been shown that the variations are tumor type- specific suggesting that SV40 has a broad host range (10, 11). Another study done on four different neuroblastoma cell lines, using primers PYV.for and PYV.rev, failed to detect SV40 Tag DNA (4). The differences in the primer sequences from the published sequence does not appear to have caused the negative results, however, since the second set of primers used in this experiment, TA1 and TA2, did not produce a positive result in the four cell lines either. The entire 309-bp sequence amplified by primers TA1 and TA2 was not read in its entirety. TA1 and TA2 were used to support the findings found using primers PYV.for and PYV.rev, and therefore only partial sequences were read which were also found to be identical to the published sequence.
The presence of SV40-like DNA in human tumors is still under investigation. Further studies are needed in order to determine whether or not the contaminated polio vaccines are the source of SV40 infection in humans. In addition, if the presence of SV40-like DNA found in the tumors of children cannot be explained by artifact or misidentification then it implies either some other source of human SV40 infection or vertical transmission from immunized subjects (12). Until the PCR techniques used to identify SV40 DNA are standardized and large scale studies are undertaken showing consistent results, the origin of SV40 infection in humans will remain elusive (12).

Acknowledgments

I would like to thank Rocco Coli and Sabrina Volpi for all their help and patience, and Dr. Rubin for the opportunity to do this project.


This document was last modified 01/31/2006.
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