Biomedical subjects

J Rommelaere

Publications and source records attributed to J Rommelaere.

At least 19 recordsLinked to original sources

PubMed1992

Nucleolin forms a specific complex with a fragment of the viral (minus) strand of minute virus of mice DNA.

Nucleolin, a major nucleolar protein, forms a specific complex with the genome (a single-stranded DNA molecule of minus polarity) of parvovirus MVMp in vitro. By means of South-western blotting experiments, we mapped the binding site to a 222-nucleotide motif within the non-structural transcription unit, referred to as NUBE (nucleolin-binding element). The specificity of the interaction was confirmed by competitive gel retardation assays. DNaseI and nuclease S1 probing showed that NUBE folds into a secondary structure, in agreement with a computer-assisted conformational prediction. The whole NUBE may be necessary for the interaction with nucleolin, as suggested by the failure of NUBE subfragments to bind the protein and by the nuclease footprinting experiments. The present work extends the previously reported ability of nucleolin to form a specific complex with ribosomal RNA, to a defined DNA substrate. Considering the tropism of MVMp DNA replication for host cell nucleoli, these data raise the possibility that nucleolin may contribute to the regulation of the parvoviral life-cycle.

Base Sequence↗

PubMed1992

In vitro infection of normal human keratinocytes by human papillomavirus type 1 followed by amplification of the viral genome in reconstructed epidermis.

Primary cultures of normal human keratinocytes were inoculated in vitro with human papillomavirus type 1 (HPV-1), the agent responsible for deep plantar warts. Upon transfer to dead de-epidermized dermis and growth at the air-liquid interface, keratinocytes reconstituted a pseudoepidermis. Under these highly differentiating conditions, HPV-1 DNA amplification was found to take place in the reconstructed epidermis, being detectable from 7 days after the transfer and persisting for at least 10 days thereafter. The extent of keratinocyte differentiation may be insufficient to allow a complete HPV infectious cycle.

Cell Differentiation↗

PubMed1992

Cooperation of oncogenes in cell transformation and sensitization to killing by the parvovirus minute virus of mice.

The established line of normal Fisher rat fibroblasts (FR3T3) is naturally resistant to the parvovirus minute virus of mice (MVM), and was used as a model system to study the influence of stepwise transformation on the susceptibility of cells to this virus. When transformed with genes encoding the class I nuclear oncoproteins large T antigen of polyomavirus (PyLT) or v-myc, cells retained a normal appearance, but acquired some ability to form colonies in soft agar. On the other hand, the class II transforming oncogenes encoding the middle T antigen of polyomavirus (PyMT) and c-Ha-ras-1 induced both morphological alterations and a high capacity for anchorage-independent growth in transfected cells. The concomitant expression of oncogenes from both classes (PyLT-(+)PyMT; v-myc+c-Ha-ras-1) induced a supertransformed phenotype characterized by the piling-up of cells into poorly adherent foci, even in low density cultures. The progressive transformation of this cellular system was found to coincide with a gradual increase in its susceptibility to MVMp (MVM prototype strain) infection. Compared to parental cells, class I, class II and double transformants proved to be sensitized to killing by MVMp to a low, moderate and large extent, respectively. Thus, oncogenes from different functional classes appeared to cooperate in the responsiveness of cells to parvovirus attack. Interestingly, this cooperation exacerbated both the killing of infected cells and their capacity to produce viral non-structural (NS) proteins, in agreement with the reported cytotoxic activity of NS polypeptides. Therefore, in this system, parameters of the parvovirus life cycle may serve as indications of the overall progression of the transformation process.

Animals↗

PubMed1992

Terminal regions of the NS-1 protein of the parvovirus minute virus of mice are involved in cytotoxicity and promoter trans inhibition.

The nonstructural (NS) transcription unit of minute virus of mice (MVMp) encodes proteins that are involved in viral DNA replication and in the regulation of homologous and heterologous promoters. Moreover, it has been shown that NS-protein accumulation is toxic for transformed cells. With the aim of identifying the NS-protein function(s) responsible for cytotoxicity, point mutations and deletions were introduced in the NS-protein-coding sequence of MVMp. This strategy indicated that in transformed human NBE cells, the NS-1 protein is indispensable for MVMp DNA replication, trans activation of the late parvoviral promoter P38, trans inhibition of the long terminal repeat promoter of the Rous sarcoma virus, and cytotoxicity. Moreover, some mutations led to the dissociation of the replicative and regulatory functions of the NS-1 protein and showed that cytotoxicity correlated with the latter, more particularly with the capacity to trans inhibit the heterologous promoter. The NS-1 sequences required for cytotoxicity were found to be restricted to the amino- and carboxy-terminal portions of the protein. Although the cytotoxicities of NS-1 extremities were weak when the extremities were tested separately, the cytotoxicities were comparable to that of the full protein when the extremities were fused. Interestingly, an overall negative charge can be predicted from the NS-1 sequence over about 100 amino acids at both ends. The conservation of this charge distribution among the NS proteins of different parvoviruses suggests that NS-1 may bear some similarities to acidic transcriptional activators.

Animals↗

PubMed1992

Transformation-dependent expression of interleukin genes delivered by a recombinant parvovirus.

The prototype strain of minute virus of mice [MVM(p)] is an autonomous parvovirus with a tropism for cells expressing a neoplastically transformed phenotype. To generate gene transfer vectors for tumor-specific gene expression, human interleukin-2 (IL-2) and murine interleukin-4 (IL-4) genes were cloned under the control of the p38 late promoter of MVM(p). Upon transfection into permissive cells, the recombinant MVMIL2 or MVMIL4 DNA was excised, amplified, and, in the presence of a helper plasmid, packaged into recombinant viral particles. The recombinant viruses were able to transfer fully functional IL-2 and IL-4 genes to permissive target cells and retained the oncotropic host range properties of the parental virus. Following infection with MVMIL2, nontransformed fibroblasts of rodent (FR3T3) or human (MRC-5) origin produced minimal IL-2 compared with the high levels of IL-2 production observed in their transformed derivatives (FREJ4 and MRC-5V1).

Cell Line, Transformed↗

PubMed1991

Post-transcriptional effect of ultraviolet light on gene expression in human cells. Stabilization of cytokine-induced and poly(I).poly(C)-induced messenger RNA.

It is well established that ultraviolet light modulates gene expression in mammalian cells, particularly at transcriptional and post-translational levels. The present study was undertaken to investigate whether the fate of mRNA is also altered in ultraviolet-light-irradiated human cells. In order to facilitate distinction between transcriptional and post-transcriptional effects, this analysis has focused on six genes whose transcription is conditional on the supply of exogenous inducers, interferon-alpha, interleukin-1 alpha or the double-stranded RNA, poly(I).poly(C). Human cells induced to express these genes were found to retain a significantly higher concentration of corresponding transcripts when irradiated with ultraviolet light at the end of the inducing treatment. This stimulation was due to dose-dependent ultraviolet-light stabilization of preformed mRNA, as shown by run-on and pulse/chase experiments. This work uncovers a new facet of the cellular response to genotoxic stresses, i.e. extension of the life-span of transcription products. Whether this stabilizing effect contributes to cell recovery by promoting gene expression remains to be determined.

Blotting, Northern↗

PubMed1991

Dose-dependent induction of resistance to terminal differentiation in x-irradiated cultures of normal human keratinocytes.

Epidermal cell clones able to proliferate under conditions that cause normal human foreskin keratinocytes (NHK) to terminally differentiate were obtained in a dose-dependent fashion after repeated x-irradiation. No terminal differentiation-resistant (TDR) clones were detected unless the total x-ray dose was split in several fractions given at protracted intervals. The x-ray-induced TDR cells were aneuploid and differed from growing NHK with regard to expression of specific protein markers of differentiation. One of the isolated TDR clones escaped senescence but failed to form tumors in nude mice. Altogether, these data indicate that NHK cultures can be used to quantitate phenotypic changes associated with neoplastic transformation of normal human epithelial cells upon exposure to defined regimens of physicochemical treatments.

Biomarkers↗

PubMed1991

Antineoplastic activity of parvoviruses.

The family of Parvoviridae is composed of small, nuclear-replicating viruses that are without envelope and contain an essentially single-stranded, linear DNA genome. Certain parvoviruses proved to have the remarkable capacity to prevent the formation of spontaneous as well as virtually- and chemically-induced tumors in laboratory animals. Established tumor cells serve as targets for the antineoplastic activity of parvoviruses, since the growth of preformed cancer cells transplanted in recipient animals can also be inhibited by these viruses. Furthermore, epidemiological studies in humans have revealed a correlation between serological evidence of parvoviral infection and a lower incidence of certain cancers. The parvoviral life-cycle appears to depend on cellular factors that are expressed as a function of proliferation and differentiation. This subordination may account for the oncotropism of parvoviruses in vivo and for the specificity of their interactions with (pre-)neoplastic cells under appropriate culture conditions. Thus, certain parvoviruses were found to preferentially lyse initiated or stably transformed cells in vitro, as a possible result of the stimulation of the production and/or activity of cytotoxic viral proteins. Parvoviruses can also have a cytostatic effect and cause the reversion of transformation traits, parallel to the down-modulation of the expression of defined genes, in particular oncogenes. Such direct disturbance of neoplastic cells or their precursors may participate in the oncosuppressive activity of parvoviruses, although indirect viral effects mediated by host defense mechanisms also deserve to be considered. Altogether, these properties suggest the possible use of parvoviruses as probes to investigate the process of malignant transformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Unscheduled DNA synthesis: a quantitative indicator of residual immunodetectable pyrimidine dimers in human fibroblasts after ultraviolet-B irradiation.

We have addressed the question whether the level of UV-B induced DNA damage can be accurately assessed by the measurement of the rate of unscheduled DNA synthesis (UDS). Cultured human fibroblasts were irradiated with UV radiation at 290, 313 or 365 nm. The LD50 was 85 J/m2 at 290 nm, 4500 J/m2 at 313 nm, and 70 kJ/m2 at 365 nm. The analysis of UDS measurements indicate complete arrest of repair processes within 24 h after irradiation, irrespective of the dose (in the range 10-60 J/m2 at 290 nm, and 250-1000 J/m2 at 313 nm). Irradiation at 365 nm failed to yield detectable evidence of UDS. Incubation of irradiated cells with an antiserum directed against both 6-4 type and cyclobutane-type pyrimidine dimers shows a clear parallelism between the disappearance of the antibody-binding determinants and the variation of the rate of UDS vs time after the end of the irradiation. Thus it is concluded that in UV-B irradiated normal cultured human fibroblasts, the lack of UDS reflects the absence of immunodetectable pyrimidine dimers.

Cell Survival↗

PubMed1991

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.

Animals↗

PubMed1990

Programmed killing of human cells by means of an inducible clone of parvoviral genes encoding non-structural proteins.

Although its dependence on the target cell type is well established, the cytopathogenicity of parvoviruses has remained elusive to date as far as its mechanism is concerned. However, indirect evidence suggested that parvoviral non-structural (NS) proteins may be the cytotoxic effectors. In order to test this hypothesis, a molecular clone of parvovirus MVMp was modified, by replacing the P4 promoter of the NS transcription unit by the glucocorticoid-inducible promoter of the mouse mammary tumour virus. Clones of neoplastic human cells that had incorporated this construct and that were induced to produce NS proteins by dexamethasone, showed a cytopathic effect and eventually died. Our data strongly suggest that the intracellular accumulation of parvoviral NS products jeopardizes the survival of the cells, which cannot be detected unless a threshold protein concentration is reached. Interestingly, a cell variant could be isolated which resisted dexamethasone-induced killing, although it was fully inducible for the production of NS proteins. This variant was also unusually resistant to infection with MVMp virions, thus confirming the essential role played by the NS proteins in the parvoviral cytotoxicity and indicating that the cytocidal activity of the parvoviral NS products is modulated by cellular factors that may vary from one cell to another.

Capsid↗

PubMed1990

NS-1 and NS-2 proteins may act synergistically in the cytopathogenicity of parvovirus MVMp.

The interaction of parvovirus minute virus of mice (prototype strain, MVMp) with simian virus 40 (SV40)-transformed human cells (NB-E) was investigated by means of transfection with MVMp molecular clones derived from the infectious recombinant plasmid (pMM984). pMM984 inhibits stable transformation of NB-E cells to geneticin resistance (G418R) upon cotransfection with the selectable pSV2neo plasmid. We show here that this inhibition is not merely caused by a repression of marker gene expression from the SV40 early region promoter in pSV2neo and rather is likely to reflect the cytotoxic action of the parvovirus. Starting from plasmid pMM984, defined mutations were introduced into the genome of MVMp and more particularly into sequences coding for the NS-1 and/or NS-2 nonstructural proteins. In this way we could show that the NS-1 protein is necessary for the inhibition of transformation to G418R and that the NS-2 protein acts synergistically to enhance this effect. Moreover, results obtained with different viral mutants indicate that the inhibitory action of NS-1 on stable transformation can be dissociated from the ability of this protein both to transactivate the parvoviral p39 promoter of the capsid protein-encoding region and to drive parvoviral DNA amplification. Altogether these data point to a probable direct toxicity of MVMp nonstructural proteins for permissive host cells.

Capsid↗

PubMed1990

Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production.

Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.

Animals↗

PubMed1990

Susceptibility of human cells to killing by the parvoviruses H-1 and minute virus of mice correlates with viral transcription.

Human fibroblasts and epithelial cells differing in their susceptibility to killing by the autonomous parvoviruses H-1 and minute virus of mice were compared for their capacity to express viral mRNAs and proteins. The transition from a parvovirus-resistant to a parvovirus-sensitive phenotype correlated with a proportional increase in the production of the three major viral transcripts and of structural and nonstructural proteins. In contrast, cell sensitization to parvovirus could not be correlated with detectable changes in virus uptake, intracellular localization of gene products, stability of viral mRNAs, or phosphorylation of viral nonstructural polypeptides. Moreover, the H-1 virus-sensitive keratinocyte line studied did not sustain a greater level of viral DNA amplification than its resistant derivative. Therefore, the differential susceptibility of the human cells tested to parvovirus infection appears to be mainly controlled at the level of transcription of the viral genome. Parvoviral gene expression could not be elevated by increasing the input multiplicity of infection in either of the cell systems analyzed. Together, these data suggest that a cellular factor(s) regulating parvoviral transcription may be modulated by oncogenic transformation or by differentiation, as both features have been shown to affect cell susceptibility to parvoviruses.

Capsid↗

PubMed1990

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.

Animals↗

PubMed1989

Inhibition by parvovirus H-1 of the formation of tumors in nude mice and colonies in vitro by transformed human mammary epithelial cells.

The formation of tumors in adult nude mice from transformed human mammary epithelial cells was drastically inhibited (greater than 80%) both after coinjection of tumoral cells and virus or after a single s.c. injection of parvovirus H-1 at the site of cell implantation prior to tumor formation. Moreover, when injected i.v. in animals bearing preformed tumors, H-1 virus was able to slow down and even in some cases to revert neoplastic growth. Thus, H-1 virus achieved the suppression of implanted tumors of human origin under conditions where the immune antitumor mechanisms of the recipient animals were dramatically impaired. Viral infection was not accompanied by detectable deleterious side effects. Imprints of H-1 virus DNA were found in one residual tumor. Normal human mammary epithelial cells were also compared with homologous transformed cells, either derived from tumors (three lines) or containing simian virus 40 (one line), for their susceptibility to the lytic replication of H-1 virus in vitro. Transformed cell lines were more sensitive to virus-induced killing than secondary cultures of normal cells. Moreover, the former had much greater abilities than the latter to amplify viral DNA and to express the viral nonstructural protein NS-1. Altogether, these results are compatible with the idea that the oncosuppressive activity exerted by H-1 virus may be mediated, at least in part, by virus replication in developing tumors.

Animals↗

PubMed1989

Identification of a transformation-sensitive nuclear protein from normal human fibroblasts that specifically interacts with minute virus of mice DNA and correlates with cell resistance to the parvovirus.

Normal human fibroblasts (MRC-5, KMS-6) were compared to transformed derivatives induced by SV40 (MRC-5V1) or gamma rays (KMST-6) for the expression of nuclear proteins that interact with the genome of minute virus of mice (MVMp), using the southwestern blot technique. A protein of 100-104 kDa apparent molecular weight was found to form a specific complex with MVMp DNA and to have an especially high affinity for the 3' terminal portion of the viral genome. This protein (p102) was differentially expressed by normal and transformed cells, i.e., its availability or DNA binding activity (or both) was much reduced in the transformants. A high level of p102 cosegregated with resistance to MVMp in cell hybrids between normal human and transformed mouse fibroblasts. Taken altogether these data suggest that the p102 protein may be a candidate for a transformation-sensitive cellular marker and for a negative regulator of parvovirus replication.

Cell Transformation, Neoplastic↗

PubMed1989

Limitations to the expression of parvoviral nonstructural proteins may determine the extent of sensitization of EJ-ras-transformed rat cells to minute virus of mice.

The FR3T3 and NRK rat cell lines and their human EJ Ha-ras-1 oncogene-transformed derivatives, termed FREJ and NREJ, were compared for their susceptibility to the parvovirus MVMp. For a similar production of p21ras protein, FREJ clones are markedly sensitized to killing by MVMp, whereas the NREJ cells are not. Such a contrasting effect of ras transformation on the sensitivity of cells of different origins to MVMp can be traced back to their respective abilities to support the parvoviral life cycle. The FR3T3 line produces a substantial amount of viral DNA whose expression in the form of the nonstructural protein NS-1 is stimulated in its transformed derivatives. Conversely, NRK cells offer an early block to parvoviral DNA replication and expression that appears to persist in the ras-transformed clones. Thus, at least two intracellular restrictions can protect normal rat cells against MVMp infection, and transformation by ras relieves one of them at the level of parvoviral gene expression. A fair correlation was also found between the degree of sensitivity of the various lines to MVMp-induced killing and their capacity to synthesize the nonstructural viral proteins, suggesting a possible role of parvoviral nonstructural proteins in cytotoxicity.

Animals↗