[Papillomaviruses and human tumors]. / Vztah papillomaviru k lidským nádorum.

The report summarizes the main results obtained in the course of our research project. The results of immunological and epidemiological studies provide further proofs that human papillomaviruses (HPV) are the etiological agents in cervical neoplasia. In addition, they raise hopes that immunological methods may be utilized in diagnostics of cervical cancer and for monitoring the clinical course of this disease in the near future. Since the etiological relationship between HPV and cervical carcinoma seems to be proven beyond reasonable doubt, the development of prophylactic and therapeutic vaccines has become the dominant of the contemporary HPV reseach. For studying immune reactions against HPV-induced tumours we developed a model of HPV16-transformed rodent cells.

Induction of anti-tumour immunity by suicide-gene-modified HPV-16-transformed hamster cells.

From K3/II, which is a highly oncogenic HPV16-transformed Syrian hamster cell line, thymidine-kinase(TK)-less cells, denoted B 49, were derived. B49 cells were transfected by a plasmid containing the herpes-simplex-virus TK gene (HSV TK) and several sub-lines expressing this gene were isolated from the transfected cultures. The HSV TK+ cells were highly sensitive to ganciclovir (GCV) and other anti-viral substances whose inhibitory effect is based on their phosphorylation by HSV TK. One of the cell lines, denoted KL1/6, exhibited relatively high stability of the HSV TK+ phenotype and was used in subsequent experiments. When KL1/6 cells were co-cultivated in the presence of GCV with various other cell lines of hamster, mouse or monkey origin, the by-stander effect (BE) was observed. GCV treatment of hamsters prevented development of tumours after the administration of KL1/6 cells but not K3/II cells. The treatment of animals with already established KL1/6-induced tumours resulted in tumour regression in all instances, but complete regression was observed only in animals carrying small tumours. The BE of KL1/6 cells on K3/II cells was also seen in vivo. In addition, concomitant immunity was observed in animals simultaneously inoculated with KL1/6 cells and K3/11 cells at 2 separate sites of the body. This effect was evident not only in animals in which KL1/6 tumours developed, but also in those in which tumour outgrowth was prevented by GCV treatment. In other experiments it was demonstrated that one KL1/6 + GCV treatment resulted in partial resistance, 2 such treatments in complete resistance to the challenge with K3/II cells.

A new cell line, GS, derived from a human renal cell carcinoma.

A new cell line, GS, of human renal cell carcinoma was established and characterized. It was derived from a metastasis of a human renal cell carcinoma, which appeared 6 years after nephrectomy. The GS cells exhibit basic characteristics of renal cell carcinoma: epithelial cell character, PAS and glycogen positivity, typical ultrastructural features. The cells have a pseudotriploid stemline with a modal number of 75 chromosomes and two marker chromosomes. GS cells formed neither colonies in soft agar nor transplantable tumours in nude mice but produced a factor(s) stimulating growth and colony forming activity of indicator cells.

Characterization of cryptovirogenic, virus-productive and helper-dependent virogenic hamster tumour cell lines.

Three hamster cell lines were established from tumours induced with the virus rescued from XC cells. The H-20 cell line produces little infectious transforming virus and synthesizes uncleaved Pr76 (product of gag gene) in the amount comparable to that obtained from PR-RSV-C-infected permissive cells. Like the helper-dependent virogenic H-18 cell line, it however produces only a marginal amount of viral glycoprotein (env gene product) as revealed by the complementation assay. Even a lower amount of the env gene product almost escaping detection by the complementation test has been found in the helper-dependent virogenic H-18 cell line. This cell line has also been shown to produce a low quantity of the gag gene product. The H-19 cell line synthesizes no detectable gag or env gene products and is not inducible by cell fusion with chicken fibroblasts even when preinfected with the replication competent td PR-C. This is in agreement with data from restriction analysis (Svoboda et al. 1983) showing that H-19 cells contain only cryptic proviral sequences represented by v-src and LTR. The three cell lines together with those described previously (Svoboda 1981) were analysed karyologically. Random changes in chromosome numbers and structural alterations in individual chromosomes were only found.

Loss of proviral sequences in RSV-transformed mouse tumour cells accompanied by the formation of double-minute chromatin bodies.

The RVA2 cell line, derived originally from a tumour induced by the Schmidt-Ruppin strain of RSV and passaged repeatedly in vivo and in vitro, was studied. This cell line was originally virogenic and the virus was rescuable with high efficiency from it. When rechecked after prolonged passage history, the efficiency of virus rescue was found to drop to very low levels and from one monocellular clone derived from such cell population no virus was rescued at all. After further 14 in vivo passages of RVA2 cells, no transforming virus rescue was obtained, even in the presence of a helper virus. Nor were the U5 sequences of LTR found in these cells. Karyologic analysis revealed the presence of double-minute chromatin bodies in the RVA2 cell population studied. The frequency of mitoses containing DMs doubled after 14 in vivo passages. The possibility is discussed that DMs as the site of gene amplification also contain amplified cellular oncogenes which might take over direction of the malignant state of the cell in which the original transforming viral gene (v-src) is no longer active or is lost.

Analysis of a cell line derived from a chicken hepatoma induced by virus MC29.

A cell line derived from a transplantable chicken hepatoma induced by virus MC29 was established. This cell line grew permanently over 2 years and could be easily recovered from frozen state. Morphology of cells did not change during the period of cultivation. Karyological analysis revealed the hypodiploid stemline with reduced numbers of microchromosomes. The cell line was not transplantable in 1-day-old chicks, but induced multiple tumour nodules in the mesenterium and liver about 8 weeks postinoculation. The cell line is virus productive. The analysis of viral proteins with the gag specific determinants showed the presence of pr76 and p110.

Stability of C-banded and C-bandless microchromosomes in clonal sublines of the RVP3 mouse tumor grown serially in vivo.

Karylogic studies were performed on three monocellular clones derived from mouse RVP3 cells, which had been originally transformed with the Prague strain of Rous sarcoma virus. All clones that had originally contained a stable number of microchromosomes, continues to retain them after prolonged passage in vivo. Centromeric heterochromatin was absent in 32% of the microchromosomes as revealed by C-banding technique. The stability of microchromosomes either positive or negative for centromeric heterochromatin is discussed in relation to double-minute chromatin bodies found in early passages of RVP3 tumor cells.

Long-term karyological study of RVP3 tumour cells grown in vitro.

The RVP3 cell line established from an in vivo passaged RVP3 tumour, which originally arose in RSV-injected mice, was analyzed at different passage levels and characterized virologically and cytogenetically. The RVP3 cell line is characterized by the presence of 2 to 5 medium-sized to large metacentric chromosomes, the majority of the mitoses contain about 1 subtelocentric chromosome, and in addition, every mitosis contains several chromosomal structures corresponding to the group called m-ms that comprises both minute chromatin bodies and microchromosomes. In three monocellular clones derived from the RVP3 cell line with a similar karyological picture it was established that the number of m-ms increased proportionally to the total number of chromosomes in individual metaphases. It was verified that: the RVP3 cell population does not contain any transmissible agent producing chromosomal changes in mouse cells; RVP3 cells are highly malignant; no RSV could be rescued from the clone tested. The significance of m-ms is discussed.

Incomplete viral genome in a non-virogenic mouse tumour cell line (RVP3) transformed by Prague strain of avian sarcoma virus.

Two cell lines, RVP3 and RVA4, derived originally from mouse tumors induced by the Prague and Schmidt-Ruppin strain of RSV, respectively, were studied. tall attempts failed to induce infectious virus production in RVP3 cells by fusion with chicken fibroblasts even if the cells were infected with avian leukosis viruses. Also, attempts to rescue the viral genome by transfection were unsuccessful. RVP3 cells harboured 31-45% of the viral genome sequences, as was shown by molecular hybridization, and therefore they were designated cryptovirogenic. The tumour cell line RVA4 did not contain any detectable viral sequences. The significance of the detection of the incomplete Rous virus genome sequences in mammalian cells is discussed.

Treatment of RSV-transformed "poorly" virogenic and non-virogenic mammalian cell lines with inhibitors of protein synthesis: failure to potentiate RSV rescue.

Treatment of the RSV-transformed "poorly" virogenic rat clones LW13-RsK4 and LW13-RsK4 R1 with cycloheximide or puromycin before fusion with chick embryo fibroblasts did not lead to RSV rescue. Neither was RSV rescued from the RSV-transformed non-virogenic mouse line RVP3 treated with 5-bromodeoxyuridine and cycloheximide or puromycin before fusion.

Evidence for animal origin of cells and rescued tumour virus from a putative human osteosarcoma cell line.

Human osteosarcoma cell lines T 1 and ZT 1 were analyzed for host origin. The results indicated that these lines must have been contaminated with rat x mouse hybrid cells. The isolated virus was identified as an avian sarcoma virus belonging to the C subgroup.