Interfacing Sca-1(pos) mesenchymal stem cells with biocompatible scaffolds with different chemical composition and geometry.

An immortalized murine mesenchymal stem cell line (mTERT-MSC) enriched for Lin(neg)/Sca-1(pos) fraction has been obtained through the transfection of MSC with murine TERT and single-cell isolation. Such cell line maintained the typical MSC self-renewal capacity and continuously expressed MSC phenotype. Moreover, mTERT-MSC retained the functional features of freshly isolated MSC in culture without evidence of senescence or spontaneous differentiation events. Thus, mTERT-MSC have been cultured onto PLA films, 30 and 100 microm PLA microbeads, and onto unpressed and pressed HYAFF-11 scaffolds. While the cells adhered preserving their morphology on PLA films, clusters of mTERT-MSC were detected on PLA beads and unpressed fibrous scaffolds. Finally, mTERT-MSC were not able to colonize the inner layers of pressed HYAFF-11. Nevertheless, such cell line displayed the ability to preserve Sca-1 expression and to retain multilineage potential when appropriately stimulated on all the scaffolds tested.

Effect of resveratrol on proliferation and telomerase activity of human colon cancer cells in vitro.

A number of studies performed in our laboratory and elsewhere, showed that resveratrol is able to prevent carcinogenesis and to impair tumor growth and progression. In order to provide additional information on the pleiotropic effects of resveratrol on malignant cells, the present study was performed to test the in vitro influence of the compound on the growth and TLMA of HT-29 and WiDr human colon cancer cell lines. The results confirmed that resveratrol has a direct, dose dependent, inhibitory effect on cell proliferation in both lines. In addition, for the first time, relatively high concentrations of this compound were found to be able to substantially down-regulate telomerase activity. These preliminary results further support the potential role of resveratrol in chemoprevention/chemotherapy of human colon tumor cells and provide the rational basis for novel strategies in cancer control.

Focus on Fotemustine.

Fotemustine is a cytotoxic alkylating agent, belonging to the group of nitrosourea family. Its mechanism of action is similar to that of other nitrosoureas, characterized by a mono-functional/bi-functional alkylating activity. Worth of consideration is the finding that the presence of high levels of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT) in cancer cells confers drug resistance. In different clinical trials Fotemustine showed a remarkable antitumor activity as single agent, and in association with other antineoplastic compounds or treatment modalities. Moreover, its toxicity is generally considered acceptable. The drug has been employed in the treatment of metastatic melanoma, and, on the basis of its pharmacokinetic properties, in brain tumors, either primitive or metastatic. Moreover, Fotemustine shows pharmacodynamic properties similar to those of mono-functional alkylating compounds (e.g. DNA methylating drugs, such as Temozolomide), that have been recently considered for the management of acute refractory leukaemia. Therefore, it is reasonable to assume that this agent could be a good candidate to play a potential role in haematological malignancies.

Drug-induced immunogenic changes of murine leukemia cells: dissociation of onset of resistance and emergence of novel immunogenicity.

n vivo exposure of tumor-bearing mice to the antineoplastic agent 5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-carboxamide (DTIC) results in increased immunogenicity of tumor cells, an event often referred to as "chemical xenogenization" (CX). To verify the hypothesis of DTIC-induced somatic mutation(s) as the major mechanism underlying CX, studies were performed to dissociate CX from the onset of drug resistance, which was invoked in the past as an event leading to selection of preexisting immunogenic clones. Therefore, experiments were done with a DTIC-susceptible tumor line treated with DTIC and quinacrine dihydrochloride (Q), an antimutagenic compound, according to selected experimental schedules. At different transplant generations, the CX and the onset of drug resistance were evaluated. The results show that a) Q does not prevent the onset of DTIC resistance, b) DTIC-resistant clones arising after treatment with DTIC plus Q are not immunogenic, and c) CX is selectively antagonized by Q. The present data confirm that DTIC-induced immunogenicity is not the result of a selection mechanism mediated by the drug and give further support to the hypothesis that the molecular mechanism of CX may be related to somatic mutation(s).

Drug-mediated antigenic changes in murine leukemia cells: antagonistic effects of quinacrine, an antimutagenic compound.

Because the antigenic changes occurring after in vivo treatment of murine lymphoma cells with 5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-carboxamide (DTIC) were suggested to result from DTIC-induced somatic mutation(s), quinacrine dihydrochloride, an antimutagenic compound, was tested for possible antagonistic activity related to this phenomenon. The increased immunogenicity of L1210Ha leukemia occurring at the first transplant generation of DTIC-treated histocompatible (BALB/cCr x DBA/2Cr)F1 male mice or the appearance of strong lymphoma-associated transplantation antigens at transplant generation 6 was prevented by simultaneous administration of quinacrine. However, the compound did not modify the antitumor or immunodepressive activity of DTIC in the mouse. We concluded that the selective antagonistic effect of quinacrine on DTIC-mediated immunogenic changes (DMIC) supported the hypothesis that the molecular mechanism of DMIC could be related to somatic mutation(s).

Antigenic changes of L5178Y lymphoma after treatment with 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide in vivo.

Immunologic alteration of the L5178Y lymphoma was obtained in vivo after treatment with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC). A single dose of 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU) "CURED" MICE CHALLENGED WITH L5178Y cells that had been treated with DIC (L5178Y/DIC) for four transplant generations; BCNU did not cure mice bearing the parent tumor. The L5178Y/DIC, treated in vivo for five transplant generations, id not grow in syngeneic mice. L5178Y/OIC cell growth and incidences of death were similar to those of parent cells when inoculated into heavily immunosuppressed mice. Adoptive transfer of lymphocytes from spleens of mice sensitized to the drug-altered tumor specifically protected immunosuppressed mice bearing the L5178Y/DIC tumor. Little protection was afforded by lymphocytes immune to the parent L5178Y tumor, whereas nonimmune lymphocytes or lymphocytes immune against unrelated tumors were completely ineffective. Anti-L5178Y/DIC lymphocytes did not cure mice challenged with the parent L5178Y tumor. Irradiated (400 R) mice previously sensitized to L5178Y/DIC cells rejected 10(2)-10(7) inocula of L5178Y/DIC cells and died when the parent L5178Y was used for challenge. It was concluded that antigeni( alterations of L5178Y cells occurred in (BALB/ctcr X DBA/2Cr)F1 mice after treatment with DIC in vivo.

Increased immunogenicity of two lymphoma lines after drug treatment of athymic (nude) mice.

Highly immunogenic sublines of L1210 and LSTRA lymphomas were obtained from athymic (nude) mice treated with 4(5)-(3,3-dimethyl-1-triazeno)imidazole-5(4)carboxamide (DIC) in vivo. Conventional mice, compatible with the parent tumor, rejected the DIC-treated sublines and were relatively resistant to a subsequent challenge with the parent lines. The DIC-treated sublines were not rejected by athymic mice, which indicated that the transplantation resistance to these tumors in conventional mice was thymus-cell dependent. In addition, there was marginal or no increase of tumor-cell immunogenicity when the parent lines were passaged in nude mice without DIC treatment. This indicated that the DIC-dependent immunogenic changes in DIC-treated leukemic conventional mice could not be ascribed merely to protection by naturally occurring antigenic clones that resulted from DIC-induced immunodepression.

Intracerebral adoptive immunotherapy of a murine lymphoma antigenically altered by drug treatment in vivo.

Intracerebral tumor neutralization assay and adoptive immunotherapy with in vivo sensitized lymphocytes were done in immunodepressed mice challenged intracerebrally with L5178Y/DTIC lymphoma, a tumor subline antigenically altered by treatment with 5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-carboxamide (DTIC) in vivo. Primary cytotoxic T-lymphocytes (CTL) were generated in vitro against L5178Y/DTIC cells with the use of splenocytes of histocompatible (BALB/cCr X DBA/2Cr)F1 donors. The extent of antitumor activity of CTL was evaluated by the measurement of tumor cell proliferation in the brain, as judged by the 125I-labeled 2'-deoxyuridine uptake values and by survival times of recipient mice. The results of the experiments showed: a) CTL were highly effective in inhibiting tumor growth when they were injected along with L5178Y/DTIC tumor cells in a Winn-type neutralization assay; b) the tumor inhibition mediated by CTL was specific, since no antilymphoma effects were detected when CTL sensitized against L5178Y/DTIC line were admixed with the parental L5178Y tumor or with other unrelated lymphoma cells; and c) local adoptive immunotherapy with CTL given on day 1, 3, or 5 after the intracerebral challenge with L5178Y/DTIC lymphoma substantially impaired tumor cell proliferation and significantly increased survival times of recipient leukemic mice.

Relationships among tumor load, route of tumor inoculation, and response to immunochemotherapy in a murine lymphoma model.

The combined effects of nonspecific immunostimulation with Candida albicans (CA) and chemotherapy were studied in (BALB/cCr X DBA/2Cr)F1 and (C57BL/6Cr X DBA/2Cr)F1 mice bearing virus-induced LSTRA lymphomas. Paradoxically, animals treated with a relatively high number of tumor cells responded better to therapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) than those challenged with a low number of tumor cells. However, the majority of mice subjected to low initial tumor load were cured when they were treated with chemotherapy or chemotherapy plus booster injection of CA at a relatively "late" stage of the disease, i.e., when high tumor load was present in tumor-bearing hosts. It has been shown that this phenomenon, provisionally called high tumor load protection, occurs when the animals are challenged ip but not when they are challenged iv with the tumor and is abolished by total-body gamma-irradiation. Moreover, marked host protection can be attained when immunostimulated mice, inoculated iv with lymphoma cells, are subjected to simultaneous challenge with high inocula of the same tumor ip, followed by BCNU administration. These data stress the importance of the peritoneal cavity for successful CA plus drug treatment and suggest that optimal tumor "antigen load" should be present at the time of CA and/or BCNU administration.

Immune inhibition of allogeneic lymphoma cells in the peritoneal cavity of mice.

Mice were sensitized with cells of normal spleen, transplantable syngeneic lymphomas, or allogeneic lymphomas differing for alloantigens specified by the major histocompatibility complex. From three to eleven days later, the allograft reactivity of these sensitized and appropriate control mice was evaluated in the peritoneal cavity by the disappearance of injected lymphoma cells or the inhibition of DNA synthesis. For the disappearance test, target cells were labeled with [125-I]-5-ido-2'-deoxyuridine before transfer. For the inhibition test, unlabeled target cells were transferred, but these cells were subsequently exposed to the DNA precursor [125-I]-5-iodo-2'-deoxyuridine. In both procedures, cells were recovered from the peritoneal cavity without killing the hosts to measure retained radioactivity. Both tests were immunogenetically specific in detecting secondary allograft reactions, but the disappearance test was less sensitive. By inhibition of DNA synthesis, it was possible to detect primary and secondary reactions, the latter three to eight days after sensitization. Alloantigens associated with the H-2K-Ir regions of Murine Linkage Group IX were more immunogenic than those associated with the Ss-H-2D-Tla regions in eliciting antilymphoma reactions, and female mice responded better than males. It was concluded that the peritoneal inhibition test is sensitive enough to monitor transplantation immunity in vivo and could be applied to animals bearing tumors in sites other than the peritoneum and undergoing chemotherapy.

Changes of the immunogenic properties of a radiation-induced mouse lymphoma following treatment with antitumor drugs.

Eight sublines of the radiation-induced lymphoma S-1033 of C57BL/10 (hereafter called B10) origin were established by exposing the cells in vivo to eight antineoplastic agents for a number of transplant generations. The parental and drug-treated sublines were tested for immunogenic properties, i.e., the ability to elicit allograft reactions in the host of origin and in congenic-resistant mice differing for the S-D or K-I-S regions of the H-2 complex. Lymphoma S-1033 and all drug-treated sublines except one were found to be essentially nonimmunogenic for B10 mice. The S-DIC subline, when exposed for 8 to 12 transplant generations to dimethyltriazenoimidazolecarboxamide, became immunogenic for syngeneic B10 mice, as judged from prolongation of survival time. Large i.v. inocula (10(7) cells) of S-1033 and of the drug-treated sublines, with the possible exception of the cyclophosphamide-treated and dimethyltriazenoimideazolecarboxamide-treated lymphomas, were more effectively rejected by K-I-S- than by S-D-incompatible mice. Dilution escape (i.e., tumor rejection after challenge with large inocula, and lethal tumor growth after injection of small inocula of lymphoma cells in allogeneic recipients) occurred in K-I-S-incompatible mice that were inoculated with S-1033 and three drug-treated (5-fluorouracil, cyclophosphamide, and pyrazocarboxamideamino) sublines. No dilution escape occurred with dimethyltriazenoimidazolecarboxamide or bischloroethylnitrosourea sublines. These data favor the hypothesis that various types of immunogenic changes of neoplastic cells may occur in tumor-bearing hosts following treatment with antineoplastic agents in vivo.

Drug-mediated increase of tumor immunogenicity in vivo for a new approach to experimental cancer immunotherapy.

In vivo treatment of leukemic mice with the antitumor agent 5-(3,3'-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) results in early increase of tumor-associated immunogenicity which is expected to evoke host-versus-graft responses. However, transplantation immunity is severely impaired in DTIC-treated mice due to the immunodepressant activity of the drug. It follows that the DTIC-mediated increase of tumor immunogenicity effect cannot be of therapeutic value in ordinary conditions. In the present report, we describe the results of studies aimed at restoring immunocompetence of DTIC-treated mice by means of adoptive transfer of syngeneic lymphoid cells. Infusion of spleen cells into DTIC-treated mice failed to restore graft responsiveness even in allogeneic tumor-host combinations. However, when DTIC treatment was followed by administration of cytotoxic alkylating agents such as cyclophosphamide (CY) or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), graft responsiveness was partially restored upon adoptive transfer of syngeneic splenocytes. (BALB/c X DBA/2) F1 (hereafter called CD2F1) mice bearing leukemia L1210 Ha were treated as follows: (a) DTIC for increasing the immunogenicity of the leukemic cells; (b) CY or BCNU; and (c) adoptive transfer of CD2F1 lymphocytes. The results showed that: (a) DTIC alone or DTIC plus spleen cells produced little or no increase in survival times with respect to untreated controls; (b) DTIC plus CY or BCNU increased survival times to a larger extent; and (c) the adoptive transfer of lymphocytes produced marked protection of leukemic mice when the hosts had been pretreated with DTIC plus CY or BCNU but not with CY or BCNU without DTIC. These data may provide a model for exploiting DTIC-induced increase of tumor immunogenicity for immunochemotherapeutic regimens.

A viral immunodepressive factor associated with experimental mouse tumors.

An immunodepressive factor (IDF) capable of inhibiting the rejection of allogeneic lymphomas in mice was detected in a number of transplantable mouse tumors. The allograft reactivity of the host was impaired for at least 10 months after IDF administration. No clinical evidence of bacterial or viral infection was detected in IDF-treated animals. Sera collected from mice at various intervals after injection of IDF showed a great increase in the IDF-titer. The IDF persisted in mouse sera for at least 3 months, whereas no IDF activity was found in sera of rats given injections of the factor. IDF was capable of replicating in vitro in mouse embryo cells, but not in rat embryo or HeLa cell cultures. IDF was inactivated in vitro by heat (65 degrees for 30 min), ultraviolet light or ether, but not by ethyl alcohol. These studies indicate that IDF is a virus capable of producing a long-lasting asymptomatic infection specifically interfering with the host's allograft reactivity. In several instances a close association between the lactic dehydrogenase virus and IDF was found. Nevertheless no conclusion was reached on the identity or nonidentity between the two viruses.

Radioresistant inhibition of lymphoma growth in congenitally athymic (nude) mice.

Several murine tumors were used to determine whether the phenomenon of tumor inhibition in athymic "nude" mice reported previously could be extended to other tumor systems in nude as well as conventional mice. The results with the L5MF-22 tumor line were confirmed, and similar data were obtained with the K36 leukemia of AKR mice and the LAF-17 leukemia of B10.A origin. This phenomenon of tumor inhibition has been called, tentatively, radioresistant inhibition of tumor and may be explained by one of several possibilities. The immunological origin of such tumor inhibition is supported by various observations. The data on tumor cell proliferation in spleens and liver of lethally irradiated mice were similar to previous findings on hemopoietic histocompatibility-incompatible lymphomas. Additionally, the nude mice were stronger responders against lymphoma cells than were conventional hosts. Another explanation is that the tumor inhibition is due to natural cytotoxicity.

Drug-mediated immunogenic changes of virus-induced leukemia in vivo.

LSTRA and RBL-5 lymphomas induced by Moloney and Rauscher leukemia viruses, respectively, were used to determine whether antigenically altered tumors induced by 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide in vivo would retain their original antigenic properties and/or have new antigenic properties. The tumors became highly immunogenic in the syngeneic hosts after 4 to 8 transplant generations with drug treatment. Syngeneic mice could be protected against challenge with the parental tumor by presensitization with the drug-altered sublines while unrelated tumor lines were incapable of protecting them. The drug-altered subline of LSTRA was used for treatment of the LSTRA in conjunction with chemotherapy, and this immunochemotherapy produced significant increases in number of survivors and increases in median survival time compared to either treatment alone. Tolerance studies indicated that there are novel antigens and parental tumor antigens associated with the drug-treated sublines.

O6-alkylguanine-DNA alkyltransferase attenuates triazene-induced cytotoxicity and tumor cell immunogenicity in murine L1210 leukemia.

Methylating and chloroethylating triazene compounds (TZCs) are effective antitumor agents in murine leukemias and can induce the appearance of novel antigens in leukemic cells (chemical xenogenization). Recently, it has been shown that TZCs might have a role in the treatment of patients affected by acute myelogenous leukemias that express low levels of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (OGAT). In this report, we have evaluated the role of this DNA repair enzyme in the leukemic cell response to the xenogenizing and cytotoxic properties of TZCs. OGAT-deficient murine leukemic L1210 cells were transfected with a recombinant ecotropic retrovirus containing the coding region for the human OGAT protein. Selected clones expressed the human OGAT transcript and had greatly increased OGAT activity. Compared to OGAT-deficient cells, OGAT-expressing cells were considerably more resistant to the xenogenizing properties of 1-(p-chlorophenyl)-3,3- dimethyl-triazene, measured in terms of leukemia graft rejection, and were less susceptible to the cytotoxic activity of the TZCs 8-carbamoyl-3-methyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one and 8-carbamoyl-3-(2-chloroethyl)imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one. These data suggest that methylation of the O6 position of guanine is involved in the appearance of increased tumor immunogenicity after exposure to methylating TZC and that OGAT is able, at least in part, to counteract the cytotoxic effects of methylating and chloroethylating agents.

Combined effects of adenovirus-mediated wild-type p53 transduction, temozolomide and poly (ADP-ribose) polymerase inhibitor in mismatch repair deficient and non-proliferating tumor cells.

Lack of p53 or mismatch repair (MR) function and scarce cell proliferation are commonly associated with tumor cell resistance to antineoplastic agents. Recently, inhibition of poly(ADP-ribose) polymerase (PARP) has been considered as a tool to overcome resistance of MR-deficient tumors to methylating agents. In the present study we demonstrated that infection with p53 expressing adenovirus (Ad-p53), enhances chemosensitivity of MR-deficient tumor cell lines to the methylating agent temozolomide (TZM), either used as single agent or, more efficiently, when combined with PARP inhibitor. Moreover, the association of Ad-p53 with drug treatment induced a more pronounced growth inhibitory effect than that provoked by Ad-p53 infection only. Cells, growth arrested by p53 transduction, and then subsequently exposed to the drugs, were still highly susceptible to cytotoxicity induced by TZM and PARP inhibitor. The results suggested that this drug combination might be effective even in non-proliferating tumor cells. It is conceivable to envisage future possible strategies to enhance cytostatic or cytotoxic effects induced by Ad-p53, based on the use of TZM, alone or combined with PARP inhibitor for the therapy of resistant tumors.

Cell-cycle progression and apoptosis in K562 and Molt-4 cells after cell-to-cell transmission of HTLV-I: modulation by interferons.

Human T-cell leukemia virus type I (HTLV-I) is mainly propagated by cell division and therefore the virus-driven proliferation of infected cells can represent a predisposing condition to final development of adult T-cell leukemia (ATL) in vivo. To correlate virus expression and cell cycle progression of recipient cells after acute infection with HTLV-I, K562 multipotent erytholeukemia and Molt-4 T-lymphoma cells were used as recipient cells in a cell-to-cell virus transmission model. Cell cycle progression was studied by flow cytometry during one duplication cycle of recipient cells and transcription of HTLV-I was evaluated during the same time course. The antiproliferative and antiviral effects of recombinant interferons alpha, beta and gamma were also evaluated on cell cycle progression and HTLV-I expression. Transcription of HTLV-I in immortalised virus-donor MT-2 T-cells was found to be related to cell cycle. After coculturing recipient K562 or Molt-4 cells with lethally irradiated, non-dividing virus-donor MT-2 cells, progression into cell cycle of recipient cells was delayed. A pre-G(1) peak, corresponding to 6-11 % apoptotic cells, was identified in cocultured Molt-4/MT-2 cells and not in Molt-4 controls, and was not affected by treatment with IFNs. Notably, no such peak was identified either in control or in cocultured K562 cells. During this time course, transcription of the viral subgenomic mRNA encoding for the env-pX region was prevalently observed. Treatment with IFNalpha and especially with IFNbeta at the onset of the cultures inhibited the growth of both control and virus-exposed recipient cells. IFNgamma was less effective. A clearcut reduction of the percentage of cells entering the S phase was observed only after treatment with IFNbeta. At the same time, in IFNbeta-treated cocultures a marked inhibition of transcription of viral mRNA was observed, suggesting that, during acute infection, treatment with IFNbeta contributes to reduce the infection of recipient cells by down-regulating both the cellular proliferation rate and virus transcription in infected cells.

Poly (ADP-ribose) polymerase inhibitor increases apoptosis and reduces necrosis induced by a DNA minor groove binding methyl sulfonate ester.

The poly(ADP-ribose) polymerase (PARP) is involved in cell recovery from DNA damage, such as methylation of N3-adenine, that activates the base excision repair process. In the present study we demonstrated that MeOSO(2)(CH(2))(2)-lexitropsin (Me-Lex), a methylating agent that almost exclusively produces N3-methyladenine, induced different modalities of cell death in human leukemic cell lines, depending on the presence of PARP inhibitor. Growth inhibition, provoked by the combination of Me-Lex and PARP inhibitor, was associated with a marked down-regulation of c-myc, increased generation of single strand breaks and apoptosis. When used as single agent, at concentrations that saturated cell repair ability, Me-Lex induced mainly cell death by necrosis. Surprisingly, addition of a PARP inhibitor enhanced apoptosis and reduced the early appearance of necrosis. Telomerase activity was completely suppressed in cells exposed to Me-Lex alone, by 24 h after treatment, whereas it did not change when Me-Lex was combined with PARP inhibitor. Thereafter, inhibition of telomerase was observed with both treatments. The results suggest new insights on different modalities of cell death induced by high levels of N3-methyladenine per se, or by the methylated base in the presence of PARP inhibitor.

Antiproliferative activity of cyclopentenone prostaglandins in early HTLV-1 infection is independent of IL-2 and is associated with HSP70 induction.

Cyclopentenone prostaglandins PGA1 and PGJ2 can inhibit the growth of HTLV-1 infected cord blood-derived human mononuclear cells (CBMC), both after acute infection and in chronically infected, immortalized cells. When CBMC were exposed to HTLV-1 infection by coculturing with lethally irradiated, virus-donor allogeneic MT-2 cells, they underwent a proliferative response, that peaked within the first week and then declined. PG treatment did not inhibit the initial proliferation (day 4) of cocultured CBMC, while multiple treatments with PGA1 and more efficiently with PGJ2, suppressed the late cell proliferation (from day 8 onward). The pharmacological effects of PGA1 and PGJ2 were reversible and therefore multiple treatments were required to maintain their antiproliferative activity. Increasing concentrations (20, 40, 80 IU/ml) of recombinant IL-2 did not affect the virus-associated proliferative response of CBMC, and exogenous IL-2 did not revert the antiproliferative effect of both PGs. Arrest of proliferation in cocultured CBMC occurred concomitantly with expression of high levels of HSP70 in the cells. In fact, though HSP70 expression was induced early (day 5) after exposure to HTLV-1, its expression was further increased after multiple PG treatments and high levels were found when the antiproliferative effect of PGs became manifest. Since HSP70 protein family is involved in the control of cell cycle as well as in antigen processing and presentation during the immune response against tumor cells and pathogens, the persistent expression of this protein in PG-treated cocultures suggested that, beside inhibiting the growth of virus-infected cells, HSP70 expression might play a role in modulating the immune function of CBMC. However, unlike in most virus infection models, in which cyclopentenone PGs exert clear antiviral effects by inhibiting the synthesis and maturation of virus proteins, no antiviral activity was found in this model of infection. This strongly suggests that the main effect of these PGs against HTLV-1 infected cells consists in inhibiting proliferation in vitro without affecting viral expression.