JC virus DNA is present in the mucosa of the human colon and in colorectal cancers

Abstract

JC virus (JCV) is a polyoma virus that commonly infects humans. We have found T antigen DNA sequences of JCV in the mucosa of normal human colons, colorectal cancers, colorectal cancer xenografts raised in nude mice, and in the human colon cancer cell line SW480. A larger number of viral copies is present in cancer cells than in non-neoplastic colon cells, and sequence microheterogeneity occurs within individual colonic mucosal specimens. The improved yield of detection after treatment with topoisomerase I suggests that the viral DNA is negatively supercoiled in the human tissues. These results indicate that JCV DNA can be found in colonic tissues, which raises the possibility that this virus may play a role in the chromosomal instability observed in colorectal carcinogenesis.

Figure 1

Schematic map of the JCV genome. Target sequences in the T antigen gene and adjacent regions were selected for amplification as described. The target sequences from nucleotide positions 4383–272 (crossing the origin of viral replication, designated 0/5130) are highlighted by the dotted line, and the nine oligonucleotide probes used to amplify the viral sequences or to hybridize the PCR products are indicated at the top. The positions of the primers and oligoprobes are as follows: ESPF, 4383–4405; JCT1, 4481–4500; 90PRO, 4521–4550; JCN1, 4573–4554; FPO, 4721–4743; JCN2, 4881–4900; ESPB, 4949–4927; JCT2, 5000–4981; JPB, 272–247. The positions of the large T and small t antigens are indicated, and the arrows indicate the direction of transcription of the early (T/t antigens) and late (agnoprotein and capsid or VP) genes.

Figure 2

Detection of the JCV T Ag sequence in human colorectal tissues. The 520-bp fragment (spanning JCV nucleotides 4481–5000, by using the primers JCT1 and JCT2) was amplified from DNA extracted from matched samples of normal colon (N) and colorectal cancers (T). (Upper) An EtdBr-stained 2% agarose gel demonstrating the 520-bp amplicons. (Lower) A Southern blot of the gel hybridized with a radiolabeled 416-bp probe for the JCV T antigen sequence obtained from the plasmid pMITC digested with HindIII. B indicates a PCR control lane (blank template plus all other reagents), and M refers to the molecular weight marker lane.

Figure 3

Effect of topoisomerase I relaxation on the amplification of JCV DNA. (A) Three matched normal-tumor pairs of DNA were selected that did not yield a 520-bp PCR product with JCT1/JCT2 primers (data not shown). After treatment with topoisomerase I, the 520-bp fragment was successfully amplified from three of the six as shown on the gel. (Top) An EtdBr-stained 2% agarose gel separation of PCR products obtained from topoisomerase I-treated templates. (Middle) A Southern blot of the same gel hybridized with radiolabeled oligoprobe FPO, confirming that three of the faintly identifiable bands on the original gel contained the authentic viral sequence. (Bottom) An EtdBr-stained 2% agarose gel of a 429-bp nested PCR product generated after 1 μl of the initial reaction was transferred to a fresh tube and amplified with the primers 90PRO/ESPB for 28 more cycles. (B) Twenty samples of colonic DNA (10 matched normal-cancer pairs), all negative for JCV sequences after one round of PCR, were treated with topoisomerase I followed by nested PCR for the 429-bp amplicon as described above. Half of the initially negative samples were found to harbor JCV sequences on three independent experiments, presented as EtdBr-stained 2% agarose gels. T, tumors; N, normal samples; M, molecular size marker; B, control (blank).

Figure 4

Semiquantitative amplification of JCV T Ag sequence from colorectal tissue samples. (Upper) The 120-bp PCR product (spanning JCV nucleotides 4881–5000) obtained by amplifying the pMad1 (JCV sequence) plasmid. Concentrations of the plasmid DNA initially were determined by A₂₆₀, and then diluted in 1 μg of human placental DNA (GIBCO), which provided an estimate of the minimal amount of template required to detect viral sequences (assuming similar efficiencies of amplification of plasmid DNA and authentic viral DNA). By using serial dilutions of template DNA to determine the point of extinction of the PCR product, the viral content in tumor specimens was estimated to be ≈0.01 viral copies/human genome equivalent. (Lower) An EtdBr-stained 2% agarose gel, and immediately below it, the corresponding Southern blot hybridized with the radiolabeled oligoprobe ESPB to confirm the sequence. Paired samples of template DNA from normal colon (N) and tumor specimens (T) from individual patients were diluted in parallel before amplification to determine the point at which the viral sequence could no longer be detected. Extinction of the PCR product from the normal samples, although still detectable from tumor samples, indicates that the tumor specimens contained a greater amount of amplifiable JCV than the matched normal colonic epithelium.

Figure 5

Heterogeneity of JCV T Ag sequences in human colorectal tissues. (Top) An EtdBr-stained 2% agarose gel demonstrating the 520-bp amplicons indicated by the arrow. A number of slowly migrating heteroduplex bands are visible in many of the lanes (T indicates a tumor specimen, N indicates a normal colonic specimen), suggesting the presence of imperfectly matched complementary sequences that form heteroduplexes that do not migrate with perfect homoduplexes. (Middle) An EtdBr-stained 2% agarose gel after the heteroduplexes were resolved by performing one additional denaturing, hybridization, and extension cycle with excess JCT1/JCT2 primers. The disappearance of the heteroduplex bands indicates that the extra bands in the top gel represented hybridization between imperfect matches of 520-bp amplicons, because the one additional amplification cycle permits copying of a perfect match for each DNA strand. If the slowly migrating bands had represented nonspecific amplification, these would not have resolved into the 520-bp band with additional amplification. (Bottom) A Southern blot derived from an electroblot of the middle gel containing resolved duplexes, hybridized with the ³²P-radiolabeled oligonucletide probe 90PRO, confirming the identity of the 520-bp band.