Anticancer effects of an oncolytic parvovirus combined with non-conventional therapeutics on pancreatic carcinoma cell lines.

Standard therapies such as surgery and chemotherapy offer only minimal improvement in pancreatic cancer. However, the viruses killing cancer cells and substances like some antibiotics and phytoalexins with anticancer potential may represent a candidate non-conventional mean of cancer treatment in the future. In this study, the effect of infection with oncolytic H-1 parvovirus (H-1PV) combined with antibiotic norfloxacin (NFX) or phytoalexin resveratrol on the survival of cell lines Panc-1 and BxPC3 derived from human pancreatic carcinoma was tested. Whereas H-1PV with NFX exerted a synergistic effect, H-1PV with resveratrol resulted in an additive effect only. All the effects were partial, but they were more pronounced in Panc-1 compared to BxPC3 cells.

Potential of tumour cells for delivering oncolytic viruses.

Autologous or allogenic tumour cells have long been used in the fight against cancer as vaccines to awaken the patient's immune system. On the other hand, oncolytic viruses have emerged in recent years as powerful therapeutic tools for selectively killing tumour cells. Yet despite recent improvements in virus production, administration and targeting, the latter strategy remains limited by poor access of oncolytic viruses to primary and metastatic tumour cells. The present review focuses on how to overcome these limitations on oncolytic virus delivery, at least in part, through the use of tumour-derived or in vitro transformed carrier cells. On the basis of existing evidence, novel strategies are proposed for using such cell vehicles, alone or in combination, both as virus factories and as anticancer vaccines.

Replication of DNA containing apurinic sites in human and mouse cells probed with parvoviruses MVM and H-1.

We studied the effect of apurinic sites on DNA replication in mouse and human cells, using parvoviruses MVM (minute virus of mice) and H-1 as probes. Although apurinic sites are efficient blocks to the replication of these single-stranded DNA viruses in vivo, depurinated parvoviruses can be reactivated if host cells have been preexposed to a subtoxic dose of UV light. The target of this conditional reactivation process is the conversion of depurinated input DNA into double-stranded replicative forms; the concomitant increase in viral mutagenesis strongly suggests that apurinic sites can be bypassed in mammalian cells.

Opposite transcriptional effects of cyclic AMP-responsive elements in confluent or p27KIP-overexpressing cells versus serum-starved or growing cells.

The minute virus of mice, an autonomous parvovirus, requires entry of host cells into the S phase of the cell cycle for its DNA to be amplified and its genes expressed. This work focuses on the P4 promoter of this parvovirus, which directs expression of the transcription unit encoding the parvoviral nonstructural polypeptides. These notably include protein NS1, necessary for the S-phase-dependent burst of parvoviral DNA amplification and gene expression. The activity of the P4 promoter is shown to be regulated in a cell cycle-dependent manner. At the G1/S-phase transition, the promoter is activated via a cis-acting DNA element which interacts with phase-specific complexes containing the cellular transcription factor E2F. It is inhibited, on the other hand, in cells arrested in G1 due to contact inhibition. This inhibitory effect is not observed in serum-starved cells. It is mediated in cis by cyclic AMP response elements (CREs). Unlike serum-starved cells, confluent cells accumulate the cyclin-dependent kinase inhibitor p27, suggesting that the switch from CRE-mediated activation to CRE-mediated repression involves the p27 protein. Accordingly, plasmid-driven overexpression of p27 causes down-modulation of promoter P4 in growing cells, depending on the presence of at least two functional CREs. No such effect is observed with two other cyclin-dependent kinase inhibitors, p16 and p21. Given the importance of P4-driven synthesis of protein NS1 in parvoviral DNA amplification and gene expression, the stringent S-phase dependency of promoter P4 is likely a major determinant of the absolute requirement of the minute virus of mice for host cell proliferation.

Transfection with extracellularly UV-damaged DNA induces human and rat cells to express a mutator phenotype towards parvovirus H-1.

Human and rat cells transfected with UV-irradiated linear double-stranded DNA from calf thymus displayed a mutator activity. This phenotype was identified by growing a lytic thermosensitive single-stranded DNA virus (parvovirus H-1) in those cells and determining viral reversion frequencies. Likewise, exogenous UV-irradiated closed circular DNAs, either double-stranded (simian virus 40) or single-stranded (phi X174), enhanced the ability of recipient cells to mutate parvovirus H-1. The magnitude of mutator activity expression increased along with the number of UV lesions present in the inoculated DNA up to a saturation level. Unirradiated DNA displayed little inducing capacity, irrespective of whether it was single or double stranded. Deprivation of a functional replication origin did not impede UV-irradiated simian virus 40 DNA from providing rat and human cells with a mutator function. Our data suggest that in mammalian cells a trans-acting mutagenic signal might be generated from UV-irradiated DNA without the necessity for damaged DNA to replicate.

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.

Selective killing of simian virus 40-transformed human fibroblasts by parvovirus H-1.

A normal strain of human foreskin fibroblasts, two SV40-transformed derivatives with finite and infinite life spans, and an established line of SV40-transformed newborn human kidney cells are compared for their susceptibility to infection with parvovirus H-1. H-1 inocula, which do not detectably alter the growth of normal cells, cause a progressive degeneration of all three SV40-transformed cultures. The resistance of normal cells is not a membrane phenomenon since they adsorb and take up H-1 as efficiently as the transformants. Moreover, the fraction of infected cells supporting the synthesis and nuclear migration of H-1 proteins is similar in normal and SV40-transformed cultures. On the other hand, the enhanced H-1 sensitivity of transformed cells correlates with a 5- to 30-fold increase in their accumulation of newly synthesized parvoviral DNA, as compared with normal cultures. This stimulation of H-1 DNA replication is most pronounced for the amplification of duplex replicative forms, although the conversion of parental single-stranded DNA to replicative forms is also enhanced to a smaller extent. In addition, SV40-transformed cells support productive H-1 infection and release a burst of infectious virus, whereas no H-1 production can be detected in the normal cell strain. The latter difference was confirmed for another series of 7 normal and 16 SV40-transformed strains of human skin fibroblasts. Altogether, these results indicate that intracellular limitations on H-1 DNA replication are associated with the abortive nature of the parvoviral life cycle in normal human fibroblasts and are overcome after SV40 transformation, resulting in the selective killing of the transformants. This observation raises the possibility that oncolysis might contribute to the oncosuppressive activity displayed by parvoviruses in vivo.

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.

Interaction of caffeine with the DNA of Chinese hamster cells.

Incubation of Chinese hamster cells with labelled caffeine leads to transfer of radioactivity to DNA. This association occurs during the S phase of the cell cycle and involves parental as well as newly synthesised strands. The replacement of thymidine by BrdUrd prevents the incorporation radioactivity from caffeine into the DNA strands containing BrdUrd. Thymine is the only base which becomes labelled and data suggesting the participation of methyl groups of caffeine in the biosynthesis of thymine are presented. Ultraviolet irradiation increases the incorporation of radioactivity from caffeine to DNA.

Induced expression of the conditionally cytotoxic herpes simplex virus thymidine kinase gene by means of a parvoviral regulatory circuit.

As a step toward the achievement of targeted expression of toxic genes, we have established a model system using the selective trans-activation of the late promoter P38 of Minute Virus of Mice (MVMp) by the parvoviral nonstructural protein NS-1. The conditionally toxic herpes simplex virus type 1 thymidine kinase (tk) gene (HSV1-tk) was cloned under the control of the P38 promoter and transfected into NIH-3T3 TK- cells. Treatment of the stably transfected cells with acyclovir (ACV) followed by infection with MVMp reduced cell survival by 3.5- to 5-fold compared to the toxic effects of ACV or MVMp alone. These results indicate that it should be possible to combine the genuine cytopathic action of parvoviruses with a specific activation of toxic genes driven by parvoviral promoters, to achieve the targeted destruction of parvovirus-expressing (in particular tumor) cells.

Highly efficient transduction and expression of cytokine genes in human tumor cells by means of autonomous parvovirus vectors; generation of antitumor responses in recipient mice.

The possible use of recombinant autonomous parvoviruses as vectors to efficiently express therapeutic cytokines in human tumor cells was evaluated in vitro and in vivo. The parvovirus H1 was used to generate recombinant viruses (rH1) that carried transgenes encoding either human interleukin 2 (IL-2) or monocyte chemotactic protein 1 (MCP-1), in replacement of part of the capsid genes. Such rH11 viruses have been shown to retain in vitro the intrinsic oncotropic properties of the parental virus. On infection with the recombinant viruses at an input multiplicity of 1 replication unit (RU) per cell, HeLa cultures were induced to release 4-10 microg of cytokine per 10(6) cells over a period of 5 days. The expression of the rH1-transduced human cytokine/chemokine could also be detected in tumor material recovered from nude mice that had been subcutaneously engrafted with in vitro-infected HeLa cells. The formation of tumors from HeLa xenografts was reduced by 90% compared with wild-type or mock-infected cells as a result of cells preinfected with IL2-expressing virus at an input multiplicity as low as 1 RU per cell. Tumors arising from HeLa cells infected with transgene-free or MCP1-expressing vectors or with wild-type H1 virus were not rejected at this virus dose. Tumors infected with rH1/IL-2 virus displayed markers indicative of their infiltration with NK cells in which the cytocidal program was activated, whereas little NK activity was detected in wild-type virus or mock-infected tumors. Altogether, these data show that the IL-2 expressing H1 vector was a more potent antineoplastic agent than the parental virus, and point to the possible application of recombinant autonomous parvoviruses toward therapy of some human tumors.

cis requirements for the efficient production of recombinant DNA vectors based on autonomous parvoviruses.

The replication of viral genomes and the production of recombinant viral vectors from infectious molecular clones of parvoviruses MVMp and H1 were greatly improved by the introduction of a consensus NS-1 nick site at the junction between the left-hand viral terminus and the plasmid DNA. Progressive deletions of up to 1600 bp in the region encoding the structural genes as well as insertions of foreign DNA in replacement of those sequences did not appreciably affect the replication ability of the recombinant H1 virus genomes. In contrast, the incorporation of these genomes into recombinant particles appeared to depend on in cis-provided structural gene sequences. Indeed, the production of H1 viral vectors by cotransfection of recombinant clones and helper plasmids providing the structural proteins (VPs) in trans, drastically decreased when more than 800 bp was removed from the VP transcription unit. Furthermore, titers of viral vectors, in which most of the VP-coding region was replaced by an equivalent-length sequence consisting of reporter cDNA and stuffer DNA, were reduced more than 50 times in comparison with recombinant vectors in which stuffer DNA was not substituted for the residual VP sequence. In addition, viral vector production was restricted by the overall size of the genome, with a mere 6% increase in DNA length leading to an approximately 10 times lower encapsidation yield. Under conditions fulfilling the above-mentioned requirements for efficient packaging, titers of virus vectors from improved recombinant molecular DNA clones amounted to 5 x 10(7) infectious units per milliliter of crude extract. These titers should allow the assessment of the therapeutic effect of recombinant parvoviruses expressing small transgenes in laboratory animals.

Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin.

The oncotropic and oncolytic behaviors of certain autonomous rodent parvoviruses make them promising vectors for anticancer gene therapies. However, these parvoviruses are often not potent enough to kill all tumor cells equally well. With the aim of enhancing the intrinsic antitumor effect and the range of natural parvoviruses, a recombinant H1 parvovirus vector was constructed that produces the Apoptin protein, a tumor cell-specific, p53-independent, Bcl-2-insensitive apoptotic effector. We compared the apoptotic activity exerted by a recombinant hH1/Apoptin virus with that of a Green Fluorescent Protein (GFP)-transducing recombinant virus, hH1/GFP, in three human tumor cell lines differing in their susceptibility to wild-type parvovirus H1-induced killing. We found that in cells that were rather resistant to the basal cytotoxic effect of wild-type H1 or the GFP recombinant virus, a parvovirus that expressed Apoptin caused a pronounced, additional cytotoxic effect. In contrast to its enhanced cytotoxicity toward tumor cells, hH1/Apoptin virus was not more toxic to normal human fibroblasts than was the wild-type H1 virus. Taken together, these data indicate that enhancing the oncotropic behavior of wild-type H1 parvoviruses with the tumor-specific apoptotic potency of Apoptin should lead to an effective replicative parvoviral vector.

Effective infection, apoptotic cell killing and gene transfer of human hepatoma cells but not primary hepatocytes by parvovirus H1 and derived vectors.

Autonomous parvoviruses preferentially replicate in and kill in vitro-transformed cells and reduce the incidence of spontaneous and implanted tumors in animals. Because of these natural oncotropic and oncolytic properties, parvoviruses deserve to be considered as potential antitumor vectors. Here, we assessed whether parvovirus H1 is able to kill human hepatoma cells by induction of apoptosis but spares primary human liver cells, and whether the former cells can efficiently be transduced by H1 virus-based vectors. Cell death, infectivity, and transgene transduction were investigated in Hep3B, HepG2, and Huh7 cells and in primary human hepatocytes with natural and recombinant H1 virus. All hepatoma cells were susceptible to H1 virus-induced cytolyis. Cell death correlated with H1 virus DNA replication, nonstructural protein expression, and with morphological features of apoptosis. H1 virus-induced apoptosis was more pronounced in p53-deleted Hep3B and p53-mutated Huh7 cells than in HepG2 cells which express wild-type p53. In Hep3B cells, apoptosis was partially inhibited by DEVD-CHO, a caspase-3 inhibitor. In contrast, H1 virus-infected primary hepatocytes were neither positive for nonstructural protein expression nor susceptible to H1 virus-induced killing. Infection with a recombinant parvovirus vector carrying the luciferase gene under control of parvovirus promoter P38 led to higher transgene activities in hepatoma cells than in the hepatocytes. Taken together, H1 virus kills human hepatoma cells at low virus multiplicity but not primary hepatocytes. Thus, recombinant H1 viruses carrying antitumor transgenes may be considered as potential therapeutic options for the treatment of hepatocellular carcinomas.

Varying sensitivity of human mammary carcinoma cells to the toxic effect of parvovirus H-1.

We have previously demonstrated lysis of non-established cultures of human mammary carcinoma cells by parvovirus H-1, which has little effect on the proliferation of corresponding normal cultures. In the present study, we examined this effect in a number of breast-tumour specimens and found them to differ as to the amplitude of their response to parvoviral attack. We first investigated whether the differences in cell sensitivity to parvovirus infection reflected the differentiation level of the initial tumour. Among the biochemical and anatomopathological indicators of original tumour differentiation, the presence of oestrogenic receptors (ER) was found to have a predictive value as to the sensitivity of derived cultures to the cytopathic effect of H-1 virus. The ER+ tumour-derived cultures showed an increased sensitivity to the lytic effect of H-1 virus compared with the ER-tumour-derived cultures, in spite of similar average proliferation rates for the two types of cultures. The proliferation rate was more heterogeneous among ER+ tumour-derived cultures and, in this group, the faster growing cultures were also the most sensitive. This observation was corroborated by the study of established cell lines retaining ER expression under in vitro culture conditions. Oestradiol was found to increase the sensitivity of these cells to the parvovirus in parallel with induction of proliferation. This effect appeared to be mediated by ER activation, since it was not observed in the ER-negative cell line MDA-MB-231. These data point to the importance of hormonal influences and cellular parameters, notably differentiation and proliferation, in determining the extent to which human cancer cells can be targets for the cytopathic effect of parvoviruses.

Are pyrimidine dimers tolerated during DNA replication of UV-irradiated parvovirus minute-virus-of-mice in mouse fibroblasts?

The replication of the single-stranded DNA of parvovirus Minute-Virus-of-MIce (MVM) was depressed when virus was exposed to UV-light prior to infection of mouse fibroblasts. Most of the viral DNA containing pyrimidine dimers was permanently blocked in its conversion to double-stranded replicative forms (RF). Yet dimers might be tolerated to a low extent, considering that a minor fraction of parental RF molecules was sensitive to the action of the UV-specific endonuclease V of bacteriophage T4, UV-irradiation of the cells prior to infection with UV-damaged MVM increased the levels of both parental RF and total viral DNA synthesized. The sensitivity of parental RF molecules to the UV-specific endonuclease was little enhanced by preirradiation of the cells and did not appear to be sufficient to account for the stimulation of RF formation in those cultures. Since parvoviral single-stranded DNA is not a substrate for nucleotidyl excision repair, one interpretation of these results would be that the process(es) activated in preirradiated cells overcome(s) barriers to viral DNA replication other than elongation blocks at pyrimidine dimers. Alternatively, pyrimidine dimers tolerated in pretreated cultures might become protected from attack by the UV-endonuclease.

Transformation of established murine fibroblasts with an activated cellular Harvey-ras oncogene or the polyoma virus middle T gene increases cell permissiveness to parvovirus minute-virus-of-mice.

Transformation of permanent rodent fibroblast cells by the polyoma virus middle T gene or the activated human Harvey-ras oncogene results in increased cellular permissiveness to the autonomous parvovirus minute-virus-of-mice. Parvoviral DNA amplification is restricted in the untransformed parental cell lines. Analysis of various parameters of the parvoviral life cycle shows that this block is partially overcome in the transformed lines.

Ultraviolet-irradiated simian virus 40 activates a mutator function in rat cells under conditions preventing viral DNA replication.

The UV-irradiated temperature-sensitive early SV40 mutant tsA209 is able to activate at the nonpermissive temperature the expression of mutator and recovery functions in rat cells. Unirradiated SV40 activates these functions only to a low extent. The expression of these mutator and recovery functions in SV40-infected cells was detected using the single-stranded DNA parvovirus H-1 as a probe. Because early SV40 mutants are defective in the initiation of viral DNA synthesis at the nonpermissive temperature, these results suggest that replication of UV-damaged DNA is not a prerequisite for the activation of mutator and recovery functions in mammalian cells. The expression of the mutator function is dose-dependent, i.e., the absolute number of UV-irradiated SV40 virions introduced per cell determines its level. Implications for the interpretation of mutation induction curves in the progeny of UV-irradiated SV40 in permissive host cells are discussed.

Susceptibility to parvovirus Minute virus of mice as a function of the degree of host cell transformation: little effect of simian virus 40 infection and phorbol ester treatment.

Transformation of mouse fibroblasts by simian virus 40 (SV40) or certain oncogenes enhances their susceptibility to the lytic replication of Minute virus of mice (MVM), a non-defective parvovirus. It was investigated whether this cytotoxic action of MVM can also be potentiated by two types of incomplete and reversible cell transformation induced either by SV40 infection or by exposure to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Although the latter two treatments trigger the expression of several traits of the transformed phenotype, they do not significantly enhance cell permissiveness to MVM.

Parvovirus H-1-induced cell death: influence of intracellular NAD consumption on the regulation of necrosis and apoptosis.

The autonomous parvovirus H-1 exerts tumor-suppressive effects in living organisms and has been shown to specifically interfere with the survival of transformed cells in culture. The mechanism(s) by which H-1 virus induces death of transformed cells is not yet well understood. It has recently been reported that H-1 virus induces apoptotic cell death in the human monocytic U937 cell line, as assessed by biochemical and morphological changes of infected cells (Rayet, B., Lopez-Guerrero, J.-A., Rommelaere, J., Dinsart, C., 1998. Induction of programmed cell death by parvovirus H-1 in U937 cells: connection with the TNFalpha signalling pathway. J. Virol. 72, 8893-8903). Here we show that parvovirus H-1 infection induced early biochemical changes pointing to apoptotic events also in the transformed human keratinocyte cell line, HeLa, and the transformed rat fibroblast cell line, P1. Morphologic changes, however, and in particular the early breakdown of plasma membrane integrity, suggested that apoptosis did not go to completion, leading to necrotic cell death as the major result of parvovirus infection of HeLa and P1 cells. Parvovirus infection of these, and to a significantly lesser extent of U937 cells, was accompanied by rapid depletion of intracellular NAD stores. Inhibition of NAD-consuming enzymes interfered with parvovirus-induced NAD depletion and increased the proportion of H-1 virus-infected cells displaying apoptotic features of cell death. In contrast, a similar prevention of NAD depletion through stimulation of NAD production had little influence on the cell death pathway, suggesting that NAD-consuming enzymes may promote necrosis in a direct way rather than through inducing the overall drop of intracellular NAD.