Simian virus 40 sequences and expression of the viral large T antigen oncoprotein in human pleomorphic adenomas of parotid glands

Abstract

Simian virus 40 (SV40) sequences of the early region coding for the large T antigen (Tag) oncoprotein were investigated in DNA samples from human pleomorphic adenoma (PA) of parotid glands. Specific SV40 sequences were detected, by PCR and filter hybridization with an internal oligoprobe, in 28 of 45 (62%) human PA specimens. None of the DNA samples from 11 normal salivary gland tissues was SV40-positive. DNA sequence analysis, carried out in all PCR amplified products from SV40-positive PA specimens, confirmed the SV40 specificity and indicated that PCR products had a sequence not distinguishable from SV40 DNA wild-type strain 776. SV40 Tag expression was revealed by immunohistochemistry with the specific monoclonal antibody Pab 101 in PA thin sections with a highly sensitive technical approach which retrieved the nuclear viral oncoprotein in 26 out of 28 (93%) samples previously found SV40-positive by PCR. Detection of SV40 sequences and Tag expression in human PA suggests that this oncogenic virus may play a role as a cofactor in the onset and/or progression of this benign neoplasm, or that SV40 DNA could replicate and express the Tag in PA cells.

Figure 1.

Agarose gels stained with ethidium bromide (dark top panels) and film images (light bottom panels) of DNA samples from human PA analyzed by snPCR and filter hybridization. MW, molecular weight markers (marker VI, Roche Diagnostics, Milan, Italy). SV40 is the PCR positive control. 1–45 are the DNA samples from PA specimens. Lane R is the negative control of the PCR reaction without DNA template. Top: PCR analysis for the SV40 Tag N-terminal sequences. Arrows indicate the product size obtained by snPCR (543 bp). Bottom: film images of snPCR amplified products of the top panels hybridized to the SV40 specific internal SV probe.

Figure 2.

Histological staining of pleomorphic adenomas for SV40 Tag oncoprotein. A (×200) and B (×320) show normal (WI 38) and SV40 Tag-transformed human fibroblasts (WI 38–13A), used as negative and positive controls, respectively. Immunoreactive cells are present only in the SV40 Tag-transformed cells. C (×140) shows cells from a normal salivary gland tissue, whereas D (×50) shows a PA of a parotid gland and the normal tissue surrounding the tumor. None of cells of the normal salivary gland is immunoreactive (C), while positive staining is present both in the transformed and normal cells of the PA sample. (D). E (×140), and F (×200) represent two different morphology, the trabecular-tubular and mixoid-stromal aspect of the PA; the positive staining appears as a dense homogeneous pattern into the nucleus of the trabecular-tubular component (E), and in the mixoid-stromal dispersed cells (F, arrows).