Hippocampal upregulation of the cyclooxygenase-2 gene following neonatal clomipramine treatment (a model of depression).

Although a putative role has been attributed to inflammation in the pathogenesis of depressive disorders, the relationship of prostaglandins, known mediators of inflammation, and depression has not been elucidated. Clomipramine is an antidepressive drug with a pro-depressive paradoxical effect in adult rats when administrated neonatally. Using this effect as a model of depression, we investigated the differential expression of the cyclooxygenase (COX-2) gene in rat brains. Rats injected neonatally with clomipramine showed depressive-like symptoms in adulthood, as well as decreased levels of the brain-derived neurotrophic factor (BDNF) and a quantitative differential expression of the COX-2 gene (Real Time PCR) and protein (immunohistochemistry) in the hippocampus. As evidenced, the relationship between a key enzyme in the prostaglandin synthesis and biological and behavioral depression-like changes opens an interesting line of investigation regarding the molecular bases of depression and its potential treatment through immunomodulatory drugs.

Mice vaccination with interleukin 12-transduced colon cancer cells potentiates rejection of syngeneic non-organ-related tumor cells.

Cell-based gene therapy after cytokine gene transfer is being investigated for autologous and allogeneic vaccination in cancer therapy. Here we show that mice vaccinated with 3-5 x 10(6) interleukin 12 (IL-12) gene-transduced CT26 colon cancer cells developed a long-lasting antitumor immune memory able to reject not only parental cells but also syngeneic, LM3 mammary, and MCE fibrosarcoma tumorigenic cells. In contrast, mice vaccinated with 0.5-1 x 10(6) CT26 cells transduced with pBabe neo IL-12 retrovirus cells (CT26-IL12) were only able to reject parental cells. An increase in the total circulating levels of IgG2a and a clear shift toward a systemic Th1 response developed, regardless of the amount of injected CT26-IL12 cells. On the contrary, a strong increase in anti-CT26-specific IgG2a levels was observed only when 3-5 x 10(6) CT26-IL12 cells were injected. Immunocompetent mice vaccinated with 3-5 x 10(6) CT26-IL12 cells developed local nodules for a few days, which then ceased growing. These nodules comprised mainly blood vessels, suggesting that an angiogenic process was taking place. CD8+ T cells were responsible for the anti-LM3 tumor cell memory, whereas CD4+ T cells were not involved. Splenocytes and lymphocytes obtained from mice immunized against CT26 cells were able to kill LM3 cells in vitro. Adoptive transfer of lymphocytes obtained from animals immunized against CT26 colon cancer cells suppressed LM3 mammary tumor growth in tumor-bearing mice. The present studies raised the possibility of isolating CTL clones and identifying CTL epitopes shared by different tumor cell types, which can be a target for cancer therapy.

IL-10 expression by CT26 colon carcinoma cells inhibits their malignant phenotype and induces a T cell-mediated tumor rejection in the context of a systemic Th2 response.

In spite of the evidence that IL-10 has Th1-immunosuppressive and anti-inflammatory effects, it has been shown that IL-10 may reduce the tumorigenic capacity of certain tumor cell types. In order to characterize the responses elicited by IL-10, we explored the effect of transducing murine CT26 colon carcinoma cells with a recombinant retrovirus expressing mIL-10. IL-10 gene transfer of CT26 cells had no effect on tumor cell growth on plastic surface but inhibited the anchorage-independent growth capacity of tumor cells and their metastatic potential as assessed by their invasive and migration ability. Expression of IL-10 also elicited an antitumor immune response involving both CD4+ and CD8+ T cells. Assessment of the immune status of the animals demonstrated that mice injected with CT26-IL10 cells showed prevalence of a systemic and tumor-specific Th2 response. Spleen cells obtained from these mice showed an increased production of IL-4 and no changes in IFNgamma levels, characteristic of a Th2 response. These results demonstrate that IL-10 affects CT26 tumor cell growth by both inhibiting the malignant phenotype and by recruiting and activating a T cell-mediated antitumor response. This T cell response occurs in the context of a shift towards a Th2 response.

Different efficacy of in vivo herpes simplex virus thymidine kinase gene transduction and ganciclovir treatment on the inhibition of tumor growth of murine and human melanoma cells and rat glioblastoma cells.

Initial studies have demonstrated the therapeutic efficacy for cancer treatment of in vivo transfer of the herpes simplex virus thymidine kinase gene followed by ganciclovir (GCV) treatment. However, recent studies have questioned the validity of this approach. Using retroviral vector-producing cells (VPC) as a source for in vivo gene transfer, we evaluated the efficacy of in vivo transduction of malignant cells using three different tumor cell models: B16 murine and IIB-MEL-LES human melanomas and a C6 rat glioblastoma. In vitro studies showed a bystander effect only in C6 cells. In vivo studies showed an inhibition of tumor growth in the two melanoma models when tumor cells were coinjected with VPC-producing retroviral vectors carrying the herpes simplex virus thymidine kinase gene, followed by GCV treatment; however, 100% of mice developed tumors in both models. Under similar experimental conditions, 70% (7 of 10) of syngeneic rats completely rejected stereotactically transferred C6 tumor cells; most of them (5 of 10) showed a prolonged survival. Treating established C6 tumors with VPC-producing retroviral vectors carrying the herpes simplex virus thymidine kinase gene and GCV led to the cure of 33% (4 of 12) of the animals. Rats that rejected tumor growth developed an antitumor immune memory, leading to a rejection of a stereotactic contralateral challenge with parental cells. The immune infiltrate, which showed the presence of T lymphocytes, macrophages, and polymorphonuclear cells at the site of the first injection and mainly T lymphocytes and macrophages at the site of tumor challenge, strengthened the importance of the immune system in achieving complete tumor rejection.

[Antitumor gene therapy using suicide genes]. / Terapia génica antitumoral mediante el uso de genes suicidas.

Tumor cells transduced with retrovirus carrying the herpes simplex-1 virus thymidine kinase (HSV-tk) are capable of transforming the antiviral drug ganciclovir (GVC) into a metabolic form only toxic to dividing cells. The efficiency of this suicide gene therapy is increased by a "bystander" effect resulting not only in the death of the recipient cell, but also in the death of non modified surrounding cells. Even though the mechanism of this "bystander" effect remains to be elucidated, strong evidence suggest that the immune system plays a main role to achieve complete tumor eradication. In the present study we evaluate the efficiency of this suicide system on three different tumor models: one human melanoma, one murine melanoma, and a rat glioblastoma. Tumors were established by injection of tumor cells s.c. in nude and C57Bl/6 mice, respectively, and stereotactically into the brain of Sprague Dawley rats. Animals in the treated group were co-injected with packaging cells producing recombinant retrovirus carrying the HSV-tk gene, and followed by i.p. administration of GVC. In short term studies, we observed inhibition of tumor growth for all the tumor models evaluated (p < 0.01). In long term studies, using the C6 rat glioma line, 50% of the animals survived longer than 75 days (p < 0.0001), and were able to reject a contralateral challenges with C6 parental cells. Histological and immunohistochemical analysis showed the presence at an inflammatory infiltrate composed by T lymphocytes, macrophages and polymorphonuclear cells. These data demonstrate that suicide genes might represent an attractive form of cancer gene therapy in the treatment of brain tumors and their intracerebral dissemination.

Suppression of SPARC expression by antisense RNA abrogates the tumorigenicity of human melanoma cells.

Acquisition of invasive/metastatic potential is a key event in tumor progression. Cell surface glycoproteins and their respective matrix ligands have been implicated in this process. Recent evidence reveals that the secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is highly expressed in different malignant tissues. The present study reports that the suppression of SPARC expression by human melanoma cells using a SPARC antisense expression vector results in a significant decrease in the in vitro adhesive and invasive capacities of tumor cells, completely abolishing their in vivo tumorigenicity. This is the first evidence that SPARC plays a key role in human melanoma invasive-metastatic phenotype development.

The expression of the secreted protein acidic and rich in cysteine (SPARC) is associated with the neoplastic progression of human melanoma.

SPARC (secreted protein acidic and rich in cysteine) is an extracellular protein associated with tissues exhibiting high rates of cell proliferation and matrix remodeling. The current work shows that the human melanoma cell lines IIB-MEL-LES, IIB-MEL-IAN, and IIB-MEL-J and different human metastatic melanomas expressed high levels of SPARC mRNA and protein. By western blot analysis we detected a single secreted 42-kDa band in human diploid fibroblasts-conditioned medium and a 45- to 40-kDa doublet in the three melanoma cell lines and all the metastatic melanomas tested. Part of the melanoma samples and cell lines showed an additional doublet of 36-34 kDa. SPARC mRNA was expressed by the three established cell lines, 14 metastatic melanoma samples, and tumors raised in nude mice, and no spliced variants were found. The heterogeneous pattern of SPARC secreted by human melanoma cells is the result of post-translational glycosylation and a specific extracellular leupeptin-inhibitable cleavage. Unlike human fibroblasts, melanoma cells did not overexpress SPARC on addition of TGF-beta. Immunohistochemical analysis showed that SPARC was strongly expressed in 100% of primary melanomas (7 of 7) and metastatic melanomas (29 of 29), moderately expressed in most of the positive dysplastic nevi (13 of 14), and only weakly expressed in nevocellular nevi (4 of 25). Normal melanocytes did not express SPARC. The data suggest that the expression of SPARC is associated with the neoplastic progression of human melanoma.

Tumor cells engineered to express interleukin-6 exhibit a reduced tumorigenicity depending on the tumor cell model.

Cytokine gene transfer to tumor cells has been demonstrated to induce tumor rejection in different murine models. However, controversial results were presented for different cytokines. In order to study the antitumorigenic activity that has been proposed for IL-6, the poorly immunogenic melanoma B16 and the colon adenocarcinoma CT26-murine cell lines, were transduced with recombinant retrovirus expressing rat IL-6. In vivo studies showed that IL-6-producing-B 16 cells inoculated s.c. in syngeneic mice, exhibited reduced tumorigenicity compared to vector-transduced B 16 cells. The histology of growing IL-6-producing tumors showed a "pseudo-nodular" pattern which correlated with a strong inhibition of the in vitro invasive capacity of these cells. IL-6-producing-B 16 cells did not develop tumors in athymic nude mice suggesting that the antitumor effect is not mediated by a normal host-T- and B-cell response. In contrast, IL-6-producing CT26 cells grew as tumors in syngeneic mice with a faster growth rate than parental and vector-transduced cells, in accordance with an increased in vitro growth kinetics. These results indicate that IL-6 expression by tumor cells demonstrate different effects depending on the tumor cell model.

[The role of SPARC gene in tumorigenic capacity of human melanoma cells]. / Rol del gen SPARC en la capacidad tumorigénica de células de melanoma humano.

Previous studies from our laboratory have demonstrated that human melanoma cell lines and tumors expressed high levels of the extracellular protein SPARC. In order to demonstrate its role in human melanoma progression, IIB-MEL-LES human melanoma cells were transfected with SPARC full length c-DNA in the antisense orientation. In vivo studies demonstrated that all the control mice injected with parental cells developed tumors, while none of the mice injected with cells obtained from three different clones with diminished levels of SPARC expression, developed tumors. These studies suggest that SPARC may play a key role in human melanoma progression.