Tissue sampling, cell line establishment and cell culture
Primary CRC resection specimens, specimens of metastases and normal tissue were received fresh from surgery, with informed written patient consent. Tissues were immediately vitally frozen in freezing medium (fetal calf serum (FCS) containing 10% DMSO) at -80°C for subsequent examinations. All cell lines were established from patient derived tumor material as previously described by [19] (used cell lines are listed in Table 1). Cell lines were cultured in T75 culture flasks using Dulbecco's Modified Eagle Medium (DMEM)/Ham´s F12 medium supplement with 10% FCS and 2mM L-glutamine as described before [20]. All procedures involving patient material were approved by the Ethics Committee of the Medical faculty, University of Rostock (reference number II HV 43/2004) in accordance with generally accepted guidelines for the use of human material.
Altogether 49 cell lines (Table 1) were tested on Polyomaviridae (SV40, JCV and BKV) and Herpesviridae (EBV). Genomic DNA (gDNA) and complementary DNA (cDNA) were used for the study (all virus preparations were a kind gift of the Institute for Medical Microbiology, Virology and Hygiene, Rostock University Medical Center and serve normally as positive controls in diagnostic virus testing). Seven of the investigated established cell lines were present in different passages (Table 1). Of six cell lines only cDNA was tested (Table 1; highlighted in grey). Primary tissue material, including normal, tumor tissue and metastases of 11 CRC patients was analyzed. Altogether 11 tissue samples of tumor material, 10 tissue samples of normal tissue and 3 tissue samples of metastases were included (Table 2).
gDNA and RNA isolation
gDNA was isolated from cell pellets using the "Wizard Genomic DNA Purification Kit" from Promega according to the manufacturer's instructions. The RNA was isolated from cell pellets using the "GeneMATRIX Universal RNA Purification Kit" from EURx according to the manufacturer’s instructions. The measurement of the isolated nucleic acids was then carried out on NanoDrop 1000 from Thermo Scientific.
cDNA Synthesis
cDNA was synthesized from RNA using the Reverase Kit (M-MuLV, RNase H minus) from Bioron (Römerberg, Germany) according to the manufacturer's instructions. The final cDNA concentration was 12.5 ng/μl after synthesis.
RT-PCR for SV40 and JC/BK
Primers used for SV40 bind to the specific SV40 nucleotide sequence for the large T antigen (PCR product size: 172bp). Used primers for JC/BK virus (Table 3) bind to the common specific JC/BK nucleotide sequence for the large T antigen (PCR product size JC/BK: 178bp and 181bp). As a positive control for SV40 gDNA, served the SV40 immortalized fibroblast cell line LCT 7. For JC/BK a positive control in form of material from an interlaboratory comparison test with proven high viral loadand provided by the Institute of Medical Microbiology, Virology and Hygiene from the Rostock University Medical Center was included. 50ng of the sample were used as start material for the PCR. Cycling conditions were as follows: 94°C – 1min followed by 40 cycles of 94°C – 30 sec, 56°C – 30 sec and 72°C – 1 min with a final extension at 72°C for 5 min.
RT-PCR for EBV
Primers used for EBV (Table 4) bind to specific EBV nucleotide sequence (PCR product size: 153bp). As a positive control for EBV gDNA served the EBV immortalized B cell line HROC212. PCR conditions were as described before.
RT-PCR for β-actin
ß-actin was used as an unregulated household gene to verify that samples are gDNA free after RNA-isolation (PCR product size: cDNA: 210bp, gDNA: 700bp). gDNA of cell line HROC111Met1 T0 M2 served as positive control for ß-actin. The annealing temperature was 58° C. PCR conditions were as described before.
Sanger Sequencing
The initial PCR product was isolated using the „High Pure PCR Product Purification Kit“ from Merck (Darmstadt, Germany) according to the manufacturer’s protocol. Next, 50ng of the isolated product were used as start material for the sequencing PCR. Cycling conditions were as follows: 94°C – 2min followed by 35 cycles of 94°C – 10 sec, 62°C – 20 sec and 72°C – 1 min with a final extension at 72°C for 5min. The sequencing reaction contained 5µl of this PCR product, 2.5µl nuclease-free water and 2.5µl primer. They were sent to a commercial sequence service provider (Eurofins, Ebersberg, Germany) which delivered sequence data back.
Agarose gel electrophoresis
To illustrate fragment sizes, a 100 bp DNA ladder (Carl Roth) was used. For electrophoresis, a voltage of 80V (15min) and 100V (45min, Elektrophoresis Power Supply, Biometra) was set. Detection took place on the BIO-Print ST4 from VILBER. Separation was performed with 2% Agarose gels in TE-buffer (reagents from Merck).
Viral infection of cell lines
To test the susceptibility of CRC cells to the JC and BK virus, HROC257 cells were infected with these viruses. The ratio of virus particles and target cells was calculated on the basis of the MOI (= multiplicity of infection) according to the following formula:
Volume (Virus) x concentration (Virus) Vv x Cv
MOI = _____________________________________ =
Volume (cell culture) x concentration (cell culture) Vc x Cc
MOI calculation for JC and BK virus:
40nm3 x 2,45x104 copies/ml
MOI = ___________________________ = 0.39
10μm3 x 2,50x102 cells/ml
500 HROC257 cells in 2 ml of DMEM were seeded on a 6-well plate and infected with virus at an MOI of 0.39. One well served as negative control. Cells were subsequently incubated at 37° C and 5% CO2.