Expression in yeast and purification of functional recombinant human poly(ADP-ribose)polymerase (PARP). Comparative pharmacological profile with that of the rat enzyme.

Human poly(ADP-ribose)polymerase (PARP) was expressed in the yeast line JEL1 under the control of a GAL promoter. Proteins were extracted and human recombinant PARP purified to apparent homogeneity. The pharmacological profile of this human enzyme was characterised in terms of the effects of known inhibitors of PARP belonging to various chemical families and this was compared with that of the rat enzyme purified from rat testes, using the same purification protocol. The rat and the human enzymes appeared very similar in terms of their sensitivities to those selected inhibitors.

F 11782, a dual inhibitor of topoisomerases I and II with an original mechanism of action in vitro, and markedly superior in vivo antitumour activity, relative to three other dual topoisomerase inhibitors, intoplicin, aclarubicin and TAS-103.

PURPOSE: F 11782 (2",3"-bis pentafluorophenoxyacetyl-4",6"-ethylidene-beta-D-glucoside of 4'-phosphate-4'-dimethylepipodophyllotoxin, di-N-methyl glucamine salt) is a newly synthesized dual catalytic inhibitor of topoisomerases I and II with major in vivo antitumour activity. In this study, we compared and contrasted F 11782 with three other known inhibitors of both these nuclear enzymes, namely aclarubicin. intoplicin and TAS-103, and established its novel mechanism of action. METHODS: In vitro growth-inhibitory effects against a panel of murine and tumour cell lines were measured by cell counting, clonogenicity or tetrazolium metabolic dye (MTT) assays. In vivo antitumour activities were evaluated against two murine tumour models (i.v. P388 leukaemia and s.c. B16 melanoma). Finally, interactions with either DNA or DNA-topoisomerases were determined using various methodologies: DNA-intercalator displacement, pBR322 DNA relaxation, kDNA decatenation, topoisomerase II extractability measurements, stabilization of topoisomerase-induced cleavable complexes (CC) in vitro and in cells, and gel retardation assays. RESULTS: F 11782 had a different profile of sensitivities and proved generally less cytotoxic than the other dual inhibitors tested in vitro, while showing significantly superior antitumour activity in vivo. F 11782, which did not stabilize CC either in vitro or in cells, was the only compound of this series capable of inhibiting the catalytic activity of both DNA-topoisomerases without interacting with DNA, and of completely impairing the binding of these nuclear proteins to DNA. Moreover, only cotreatment of cells in vitro with F 11782 enhanced the cytotoxic activity of etoposide. CONCLUSION: These results emphasize the novel mechanism of action of F 11782 vis-a-vis the other dual inhibitors of topoisomerases I and II and so augur well for its future clinical development.

F 11782, a novel epipodophylloid non-intercalating dual catalytic inhibitor of topoisomerases I and II with an original mechanism of action.

F 11782, a novel epipodophylloid, proved a potent inhibitor of the catalytic activities of both topoisomerases I and II. Unlike classical inhibitors such as camptothecin or etoposide, F 11782 did not stabilise cleavable complexes induced by either topoisomerases I or II nor did it preferentially inhibit the religation step of the catalytic cycle of either enzyme. F 11782 neither intercalated DNA nor bound in its minor groove, and showed only weak inhibition of the ATPase activity associated with topoisomerase II. F 11782 appeared to act by inhibiting the binding of topoisomerases I and II to DNA in a manner dependent both on drug and enzyme concentrations, via a mechanism not previously described or shared by other known topoisomerase 'poisons' or inhibitors. In contrast, F 11782 had only a weak effect or none at all on various other DNA-interacting enzymes. In conclusion, F 11782, as a non-intercalating, specific catalytic inhibitor of both topoisomerases I and II with an original mechanism of action, may be considered to represent the first of a new class of topoisomerase-interacting agents.

Characterization of the biological and biochemical activities of F 11782 and the bisdioxopiperazines, ICRF-187 and ICRF-193, two types of topoisomerase II catalytic inhibitors with distinctive mechanisms of action.

F 11782 is a newly identified catalytic inhibitor of topoisomerases I and II, without any detectable interaction with DNA. This study aimed to establish whether its catalytic inhibition of topoisomerase II was mediated by mechanisms similar to those identified for the bisdioxopiperazines. In vitro combinations of F 11782 with etoposide resulted in greater than additive cytotoxicity in L1210 cells, contrasting with marked antagonism for combinations of etoposide with either ICRF-187 or ICRF-193. All three compounds caused a G2/M blockade of P388 cells after an 18-h incubation, but by 40 h polyploidization was evident only with the bisdioxopiperazines. Gel retardation data revealed that only F 11782, and not the bisdioxopiperazines, was capable of completely inhibiting the DNA-binding activity of topoisomerase II, confirming its novel mechanism of action. Furthermore, unlike ICRF-187 and ICRF-193, the cytotoxicity of F 11782 appeared mediated, at least partially, by DNA damage induction in cultured GCT27 human teratoma cells, as judged by a fluorescence-enhancement assay and monitoring p53 activation. Finally, the major in vivo antitumor activity of F 11782 against the murine P388 leukemia (i.v. implanted) and the B16 melanoma (s.c. grafted) contrasted with the bisdioxopiperazines' general lack of activity. Overall, F 11782 and the bisdioxopiperazines appear to function as quite distinctive catalytic topoisomerase II inhibitors.

Differential expression of topoisomerase I and RAD52 protein in yeast reveals new facets of the mechanism of action of bisdioxopiperazine compounds.

A screening procedure which permits identification of compounds based on their activities against specific biological targets directly in a living organism, Saccharomyces cerevisiae, has been established as part of our new drug discovery programme. Use of this assay has provided the first direct evidence that TOP1 and RAD52 proteins are involved in the mode of action of bisdioxopiperazine ICRF compounds, which thus express a mode of action quite distinctive from the other known TOP2 inhibitors evaluated. The functional assay is based on a comparison of pairs of yeast differing in their phenotypes by specific traits: the expression or lack of expression of ectopic human DNA topoisomerase I, with or without that of the RAD52 gene. Amongst a series of anticancer agents, inhibitors of topoisomerase I (camptothecin) were identified as such in yeast expressing human topoisomerase I, whilst the presence or absence of RAD52 protein permitted the discrimination of compounds generating double-stranded DNA breaks, either directly (bleomycin) or involving DNA adduct formation (cisplatin), or indirectly with DNA damage mediated via inhibition of the topoisomerase II enzyme (etoposide). Notably, however, both the RAD52 protein and the lack of TOP1 enzyme appeared implicated in the cytotoxic activities of the series of bisdioxopiperazine ICRF compounds tested. This functional assay in a living organism therefore appears to provide a valuable tool for probing distinctive and specific mode(s) of action of diverse anticancer agents.

Differential in vitro interactions of a series of clinically useful topoisomerase-interacting compounds with the cleavage/religation activity of the human topoisomerase IIalpha and IIbeta isoforms.

The topoisomerase II (TOP2)-associated DNA cleavage activity and the DNA sequence preference of 20 antitumor drugs, including 15 TOP2-interacting compounds, have been defined. Four major classes of drugs have been identified: (i) those which enhanced the stabilization of cleavable complexes at a single major site (e.g. amsacrine, doxorubicin), or (ii) at many sites (e.g. etoposide, azatoxin), with chemically related compounds having very similar, although not identical, cleavage patterns (e.g. etoposide, GL331 and Top-53); (iii) those which inhibited DNA breakage (e.g. aclarubicin, actinomycin D); and (iv) those which did not visibly interfere with TOP2-mediated cleavable complexes (e.g. ICRF-187, camptothecin). All drugs tested induced similar overall patterns of sites of preferred DNA cleavage, in the presence either of the two known isoforms, TOP2alpha or TOP2beta, although relative intensities of signals at each position varied. It has been further shown that etoposide and its derivatives blocked the religation step downstream of the DNA cleavage step, whereas amsacrine, ellipticine, azatoxin and genistein acted upstream through enhancement of DNA cleavage. The information provided by this mechanistically based comparison can now be exploited in designing or synthesizing novel TOP2-interacting agents.

Differential sensitivities of recombinant human topoisomerase IIalpha and beta to various classes of topoisomerase II-interacting agents.

A series of topoisomerase-interacting antitumour agents were tested for their ability to differentially inhibit the catalytic activity of either topoisomerase (TOPO) IIalpha or beta, as judged by a DNA decatenation assay. The alpha form, relative to the beta isoform, proved 1 to 3 times more sensitive to nonintercalating complex-stabilizing TOPO II-interacting agents (etoposide and derivatives) and up to 18 times more sensitive to non-complex-stabilizing inhibitors of TOPO II ((+/-)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane [ICRF 159] and meso-2,3-bis(3,5-dioxopiperazine-1-yl)butane [ICRF 193]). However, the beta form of the enzyme appeared 1 to 3 times more sensitive to intercalating TOPO II-interacting agents (daunorubicin, aclarubicin and mitoxantrone). A possible implication of these data are that tumours preferentially expressing either the alpha or the beta isoform may be differentially responsive to various classes of TOPO II-interacting agents.

Yeast cells expressing differential levels of human or yeast DNA topoisomerase II: a potent tool for identification and characterization of topoisomerase II-targeting antitumour agents.

PURPOSE: To identify and characterize the specificity and potency of topoisomerase II-interacting antitumour drugs in an in vivo model utilizing the yeast Saccharomyces cerevisiae. METHODS: Four yeast transformants were selected for the expression of either human or yeast DNA topoisomerase II at different, biologically relevant, levels under the tight control of promoters of various strengths. RESULTS: Analyses of 24 drugs permitted their classification into three distinct groups, depending on whether they induced topoisomerase II-related cytotoxicity (etoposide), showed nonspecific cytotoxicity (camptothecin), or exerted no cytotoxicity at all (vinorelbine). Within the first group different patterns of action were distinguishable: (1) classical topoisomerase II expression-dependent cytotoxicity for both species of enzyme (e.g. etoposide, amsacrine, doxorubicin, actinomycin D), although amsacrine and TOP 53 were more active, respectively, on human and yeast topoisomerase II; and (2) compounds that appeared to poison only one species of topoisomerase II with, for example, genistein and the bisdioxopiperazine ICRF-193 lethally targeting only the human type, and mitoxantrone only the yeast isozyme. Three of the 16 known topoisomerase II inhibitors tested were not correctly identified with this assay, possibly owing to restricted cell wall permeability or to the absence of correct processing pathways such as, for example, in the case of the prodrug etopophos. CONCLUSION: This methodology, in vivo in yeast, selected for a large range of potent topoisomerase II-targeting anticancer agents. Of particular interest in this yeast model, and in contrast to yeast topoisomerase II, human topoisomerase II was shown to confer dominant sensitivity in the presence of the series of bisdioxopiperazine derivatives tested. This assay therefore has the potential easily to identify and characterize the potency and specificity of synthesized anticancer drugs with modified original chemical structures or those present, for example, in natural plant extracts or marine organisms.

MTS1 expression and prognosis in acute myeloid leukemia.

MTS1, a tumor suppressor gene located on chromosome 9p21, has been shown to be altered in a number of human tumor cell lines, primary solid tumors, and leukemias. In this study we found low expression of MTS1 in lymphocytes from seven of nine healthy donors, but in none of eight granulocyte samples from the same controls, suggesting a physiological role of MTS1 in peripheral blood cells capable of proliferation, but not in end-stage differentiated cells. We detected MTS1 mRNA expression in 38 of 57 patients (66%) with acute myelogenous leukemia (AML) treated in a standardized clinical protocol. No deletion of the MTS1 gene was found in any of the AML samples tested. There was no significant association between expression of MTS1 and response to therapy, progression-free, or overall survival. Except for a negative correlation between MTS1 level and leukocyte count at diagnosis (p = 0.03), there was also no association with any of the known prognostic parameters in AML. We conclude that MTS1 shows a significantly higher expression in leukemic than in normal peripheral blood cells, that deletion of MTS1 is not a frequent event in AML, and that its expression is not significantly correlated with outcome of the disease.

Five putative drug resistance parameters (MDR1/P-glycoprotein, MDR-associated protein, glutathione-S-transferase, bcl-2 and topoisomerase IIalpha) in 57 newly diagnosed acute myeloid leukaemias. Swiss Group for Clinical Cancer Research (SAKK).

UNLABELLED: Using a modified quantitative reverse transcriptase (RT) PCR assay in 57 patients with acute myeloid leukaemia (AML) from a Swiss Phase III multicentre study (SAKK 30/85), we measured the m-RNA expression of the genes from the multidrug resistance gene 1 (MDR1), the multidrug resistance associated protein (MRP), glutathione-S-transferase (GST) pi, bcl-2 and topoisomerase (topo) IIalpha. P-glycoprotein (p-gp) was measured by Western blot, and GST activity by functional assays. To analyse progression-free (PFS) and overall survival (OS), parameters were prospectively divided into "low" and "high" groups, according to their median values (exceptions: MDR1 and p-gp). Median follow-up was 60 months. RESULTS: MDR1- and MRP mRNA levels correlated with each other (r=0.54, Spearman), FABM4/M5 and extramedullary disease. "Low" bcl-2-mRNA predicted longer PFS: 22 months vs. 7 months (median,p=0.02, log rank), and longer OS: 64 months vs. 14 months (p=0.06). "Low" topo IIalpha predicted poorer outcome: median PFS 9 vs. 19 months (p=0.03); median survival 12 months vs. "not reached" (p=0.03). An improved outcome tendency, albeit nonsignificant, was seen in p-gp-negative patients. In a Cox model adjusted for age, performance status, presence of Auer rods, FAB type and clinical response, bcl-2 and topo IIalpha mRNA levels retained their predictive values.

mRNA expression, measured by quantitative reverse transcriptase polymerase chain reaction, of five putative drug resistance parameters, in normal and leukaemic peripheral blood and bone marrow.

Using a quantitative reverse transcriptase PCR assay, the mRNA expression of five putative drug resistance-related genes were assessed in normal peripheral (n = 14) and bone marrow (n = 4) mononuclear cells from healthy donors and patients with acute myeloid leukaemia (n = 11). The mRNA levels of MDR1, the multidrug resistance-associated protein and glutathione-S-transferase pi were equally expressed in both compartments. Bcl-2 mRNA was slightly higher in the leukaemic marrow samples. However, topoisomerase II alpha mRNA levels were found to be much higher in normal and leukaemic marrow cells compared to peripheral blood (p < 0.01), which may, in part, reflect the different proliferation pattern of the mononuclear cells in the two compartments. Such findings could be important for researchers using bulk assays in a mix of samples from peripheral blood or bone marrow to investigate prognostic factors in patients with leukaemia.

Assessment of drug-induced dysregulations among seven resistance-associated genes in human tumour cell lines.

Drug-resistance in cell lines and in malignant human tumours is associated with dysregulation of several genes including mdr1, MRP1, GST-pi, bcl-2, DNA topoisomerase II alpha and beta, and thymidine kinase I. mRNA expression was evaluated by quantitative RT-PCR coupled with HPLC in three human tumour cell lines and drug-resistant (DR)-sublines. DR sublines from RPMI-8226 and KB cells specifically overexpressed the mdr1 gene without major changes observed in other putative DR-associated genes. In contrast, the DR-H69 cells exhibited a 34-fold overexpression of the MRP gene accompanied by significant down-regulation of both DNA topoisomerase II alpha and bcl-2 mRNA gene expression, by factors of 43 and 13 respectively. These results demonstrate the concomitant down regulation of topoisomerase II alpha and bcl-2 genes in response to DR. Furthermore, differential patterns of gene dysregulations appear to vary depending upon both the drug used to select resistance and cellular origin.

The ubiquitous VA68 isoform of subunit A of the vacuolar H(+)-ATPase is highly expressed in human osteoclasts.

The 67-kDa subunit A of the vacuolar-type H(+)-ATPase carries the high affinity ATP binding site and together with the 57-kDa subunit B forms the catalytic domain. Two isoforms of subunit A, VA68 and HO68, were cloned from an osteoclastoma cDNA library. We have analyzed their respective expression patterns in different tissues by RNAase A protection and in situ hybridization. The HO68 isoform was found to be present only in the tumor originally used to construct the cDNA library, whereas the ubiquitous VA68 RNA isoform was detected in large osteoclastic cells, as well as in brian and kidney, by RNAse protection assay. Furthermore, we localized the strong signal observed in osteoclastoma RNA to the large osteoclastic cell by in situ hybridisation. These findings suggest that the subunit A highly expressed in human osteoclasts is the ubiquitous isoform, VA68.

Nonradioactive quantification of mdr1 mRNA by polymerase chain reaction amplification coupled with HPLC.

We describe a new strategy for quantifying mRNA by using polymerase chain reaction (PCR) coupled with HPLC. PCR-amplified products are directly analyzed with a specific HPLC column and are quantified by standardization against a housekeeping gene, beta-actin. To evaluate the experimental conditions, we examined the multidrug-resistance-associated mdr1 gene expression in two drug-sensitive cell lines (RPMI-8226 and KB-3-1) and in their drug-resistant sublines. This revealed a significant overexpression of the mdr1 gene in chemoresistant sublines that paralleled the degree of in vitro drug resistance and the amounts of mRNA determined by other methods. Results were consistently reproducible, with imprecision (CV) < 25%. The reverse transcription option PCR/HPLC protocol described here is a sensitive, nonradioactive method for quantifying mdr1 mRNA in cell lines or clinical tumor samples, and can be applied to the simultaneous quantification of several mRNA species.

Heterogeneity of vacuolar H(+)-ATPase: differential expression of two human subunit B isoforms.

The catalytic domain of the vacuolar proton ATPase is composed of a hexamer of three A subunits and three B subunits. Here we describe the cloning and characterization of a cDNA isoform of subunit B, HO57, from an osteoclastoma cDNA library. HO57 is represented by three species of mRNA of 1.6, 2.6 and 2.8 kb and is expressed at low levels in a range of human tissues, but at significantly higher levels in brain, kidney and osteoclastoma, and is probably an ubiquitously expressed isoform. In contrast, the kidney-specific isoform has an mRNA of 2 kb and is specifically expressed at high levels only in kidney and, at a lower level, in placenta. Thus the HO57 isoform is integral to the vacuolar ATPase found in the general secretory system of all cells as well as in vacuolar-ATPase-rich sources such as neurones and osteoclasts, whereas both the kidney-specific isoform and HO57 are highly expressed in the kidney. Furthermore, we show by in situ hybridization that HO57 is the only isoform that is exclusively and highly expressed by osteoclasts.

Cloning and tissue distribution of subunits C, D, and E of the human vacuolar H(+)-ATPase.

The vacuolar proton ATPase (V-ATPase) translocates protons into intracellular organelles or across the plasma membrane of specialised cells such as osteoclast and renal intercalated cells. The catalytic site of the V-ATPase consists of a hexamer of three A subunits and three B subunits which bind and hydrolyse ATP and are regulated by accessory subunits C, D and E. cDNAs encoding subunits C, D, and E were cloned from human osteoclastoma, a tissue highly enriched in osteoclasts, as a first step in the characterisation of the V-ATPase used by the osteoclast. By Northern blot analysis only one mRNA species were detected for each of these subunits, which is consistent the constant transcription level in all tissues irrespective of the presence of specialised cells highly enriched in V-ATPases.

Identification of two subunit A isoforms of the vacuolar H(+)-ATPase in human osteoclastoma.

Subunit A is thought to be the main component of the catalytic site of the vacuolar-type H(+)-ATPase. Screening of a cDNA library made from human osteoclastoma tumor tissue revealed the presence of two isoforms of subunit A. HO68 is a cDNA of 3.1 kilobase pairs, corresponding to a mRNA of approximately 3.4 kilobases in osteoclastoma only, encoding a protein of 615 amino acids with a predicted molecular mass of 68177 Da. A second subtype, VA68, corresponding to a mRNA of approximately 4.8 kilobases was present in all tissues analyzed, and codes for a predicted protein of 617 residues and theoretical molecular mass of 68264 Da. These clones share homology with previously published subunit A sequences, and this, together with the tissue distribution of the mRNA, suggests there are ubiquitous (VA68-type) and tissue-specific (HO68-type) isoforms. HO68 shows the closest sequence homology (95% at the amino acid level) to subunit A of a proton-secreting vacuolar-type H(+)-ATPase located in the apical membrane of midgut goblet cells of tobacco hornworm larva (Manduca sexta). We propose that HO68 could correspond to an isoform of subunit A specific for a vacuolar-type H(+)-ATPase located in the osteoclast plasma membrane.

The c-Ha-ras oncogene induces increased expression of beta-galactoside alpha-2, 6-sialyltransferase in rat fibroblast (FR3T3) cells.

Alteration in cell surface carbohydrates, and in particular cell surface sialylation, have been known to occur during oncogenic transformation. To examine the basis for such changes, we have transformed the rat fibroblast cell line FR3T3 with the oncogenes c-Ha-ras EJ, v-mycOK10, v-src, polyoma virus middle T or the transforming bovine papilloma virus 1 (BPV1), and measured the sialytransferase activities of cellular lysates. We found that, in contrast to all other oncogenes examined, c-Ha-ras induced a striking increase in beta-galactoside alpha-2,6-sialytransferase (Gal alpha-2,6-ST) activity in FR3T3 cells. This increase in Gal alpha-2,6-ST activity resulted in the increased expression of cell surface alpha-2,6-linked sialic acid on cell surface glycoconjugates, as determined by cell staining with fluorescein-labelled Sambucus nigra agglutinin. Immunoprecipitation and immunofluorescence experiments revealed that the increase in Gal alpha-2,6-ST activity was due to an elevation of expression of the enzyme. Moreover, Northern analysis suggested that the increased expression of this enzyme was the result of an increase in the steady-state mRNA level of the Gal alpha-2,6-ST gene. These results support the notion that alterations seen in cell surface glycoconjugates during oncogenic transformation can be the result of altered expression of glycosyltransferases.

Initiation of transcription from the minute virus of mice P4 promoter is stimulated in rat cells expressing a c-Ha-ras oncogene.

Transformation of FR3T3 rat fibroblasts by a c-Ha-ras oncogene but not by bovine papillomavirus type 1 is associated with an increase in the abundance of mRNAs from prototype strain MVMp of infecting minute virus of mice, an oncosuppressive parvovirus. This differential parvovirus gene expression correlates with the reported sensitization of ras- but not bovine papillomavirus type 1-transformed cells to the killing effect of MVMp (N. Salomé, B. van Hille, N. Duponchel, G. Meneguzzi, F. Cuzin, J. Rommelaere, and J. Cornelis, Oncogene 5:123-130, 1990). Experiments were performed to determine at which level parvovirus expression is up-regulated in ras transformants. An MVMp "attenuation" sequence responsible for the premature arrest of RNA elongation was either placed or not placed in front of the chloramphenicol acetyltransferase gene and brought under the control of MVMp early promoter P4. Although the MVMp attenuator reduced P4-driven chloramphenicol acetyltransferase expression, the extent of attenuation was similar in normal and ras-transformed cells. Moreover, the analysis of P4-directed viral RNAs in MVMp-infected cultures by RNase protection and nuclear run-on assays also revealed a transcription elongation block of a similar amplitude in both types of cells. In addition, the stabilities of the three major parvoviral mRNAs did not vary significantly between normal and ras-transformed cells. Hence, it is concluded that the ras-induced increase in the accumulation of parvoviral mRNAs is mainly controlled at the level of transcription. Consistently, the TATA motif of the P4 promoter proved to have a differential photoreactivity when tested by in vivo UV footprinting assays in ras-transformed versus normal cells.

Sensitization of transformed rat cells to parvovirus MVMp is restricted to specific oncogenes.

The rat cell line FR3T3 was transformed with the retroviral oncogenes v-myc or v-src, with the DNA tumor viruses SV40 or bovine papilloma virus strain 1 (BPV-1) or with the 69% transforming region of BPV-1. The transformants were compared with the uncloned parental line for their susceptibility to the lytic effect and to the replication of MVMp, an autonomous parvovirus. Expression of v-myc and v-src proteins and of SV40 large T antigen correlated with a greater cell susceptibility to MVMp-induced killing. Thus, the expression of both cytoplasmic and nuclear oncogene products may sensitize rat fibroblasts to MVMp. In contrast, cell lines transformed by BPV-1, including highly tumorigenic and tumor-derived clones, were on the average as resistant as the parental cell line to MVMp infection. A similar resistance to MVMp-induced killing was displayed by BPV-1-transformed NIH3T3 cells. However, supertransformation of one of the BPV-1-transformants by the human EJ-Harvey ras-1 oncogene, known to sensitize FR3T3 and NIH3T3 cells, correlated with an increase in susceptibility to MVMp. Therefore, the failure of BPV-1 transformation to sensitize murine cells to parvoviral attack may be ascribed to the tumor virus rather than to the cells undergoing transformation. Hence, cell sensitization to MVMp appears to be oncogene-specific and cannot be taken as an absolute correlative with neoplastic transformation.