Primary aim of this study was the detection of the potentially oncogenic viruses SV40, JCV, BKV and EBV in patient-individual low passage (<P50) CRC cell lines [21]. Therefore, gDNA and cDNA from established and characterized cell lines and corresponding patient tumor (Tu) and normal (N) tissues, as well as metastases (Met) were examined by RT-PCR.
Analysis of SV40 in cell lines and primary tissue samples
For the detection of SV40, altogether 49 individual patient-derived colorectal tumor cell lines were examined. No specific band pattern could be shown for the SV40 virus in all cell lines (Figure 1).
The positive control shows a clear specific band at 172bp. In order to clarify whether SV40 sequences are detectable at RNA level, additionally cDNA from the same cell lines was examined. Again, no viral sequences could be amplified with the positive control showing a band at 172bp (not shown). However, examination of primary tumor material revealed signals in HROC277Tu and HROC296Tu (Figure 2).
PCR product sizes were about 170bp and 300bp, respectively. To clarify the specificity of the detected bands, a reamplification of the PCR products was carried out with the same primers. However, the initially detected band of HROC296 (300bp) could not be reamplified, while the reamplification product of HROC277 exhibited an unspecific smear and must therefore also be regarded as negative (Figure 3).
Analysis of JC and BK virus in cell lines and primary tissue samples
In 2 of 49 examined cell lines, the PCR with primers designed for the amplification of JCV/BKV sequences delivered a signal at approximately 180bp (HROC348Met P9 and HROC212 P17) (Figure 4) suggesting a viral infection. Again, the positive controls delivered the expected products of 178bp (JCV) and 181bp (BKV), respectively (Figures 4 and 5).
To verify these findings, a highly sensitive diagnostic qPCR (TaqMan®) was performed to detect JCV and BKV at the Institute of Microbiology of the University Medical Center, Rostock as described originally by Watzinger et al., in 2004 with minor modifications [22]. However, no JC or BK viral load could be detected in any of the samples and the initial results could thus not be confirmed. In analogy to the SV40 analysis, cDNA was also assayed for JCV and BKV. All cDNA samples were negative (data not shown).
The examination of primary tumor material revealed a signal at approximately 380bp for the samples HROC107 N/Tu, HROC126 N, HROC183 N/Tu and HROC212 N (Figure 5). As product size was greater than the expected 178bp or 181bp for JC/BK virus, a reamplification PCR was performed with the PCR product of the primary materials. Here, the result of the first RT-PCR could be confirmed. All samples of the reamplification showed a product size of approximately 380bp. To check the specificity of this amplified nucleotide sequence of the primary patient materials, the reamplified amplicons were sequenced by Sanger sequencing. This analysis identified a human DNA sequence from clone RP11-203L2 (chromosomes 9q21.11-21.2) with a 99% identification probability. Thus, in none of the investigated primary materials, JCV or BKV were detectable.
Analysis of EBV in cell lines and primary tissue samples
RT-PCR revealed several amplification products at about 700bp in size in the examined cell lines and primary tissue samples with no difference between tumor and normal tissue (data not shown). The product size is about 700bp and is thus by far larger than the expected product size of 150bp. A reamplification failed to reproduce the initial 700bp bands in all cases. Similar to the observation for SV40, either an unspecific smear or no signal at all was obtained. In conclusion, it can be assumed that the 700bp amplicons are non-specific.
Susceptibility of HROC257 to JC and BK virus
To test the susceptibility of CRC cells to infection with JC and BK virus, the cell line HROC257 was infected with JC and BK viruses with an MOI of 0.39 as explained before. Growth and morphology of the cells were documented photographically (Figure 6). Particular attention was paid to the following parameters: changes in growth behavior / inhibition of proliferation, cell lysis or potential transformatory effects, reflected in the morphology of the cells. During cultivation of the HROC257 cells in the presence of the JCV and BKV, no morphological changes could be observed in comparison to untreated control cells. As shown in Figure 6, the growth behavior of infected and uninfected cells is similar. In order to further analyze susceptibility to JCV and BKV, these HROC257 cells were subjected to the above described RT-PCR, 40 days after viral infection. Both, cell culture supernatant and isolated cellular gDNA were used as templates. In positive controls, the analysis of cell culture supernatant showed bands at 178bp (JCV) and 181bp (BKV). But no products were detectable in supernatant of neither JCV nor BKV treated cells (data not shown). This result suggests that either no or very few viral particles were present in the supernatant. In addition, isolated cellular gDNA of virus-treated HROC257 cells were tested for JCV and BKV after 40 days of incubation. But again no viral amplicon could be detected (data not shown). These results indicate that HROC257 were not susceptible to JC or BK virus infection.