Expression of high affinity choline transporter during mouse development in vivo and its upregulation by NGF and BMP-4 in vitro.

An important feature of cholinergic neurons is high-affinity choline transport, which allows them to reuse choline for the synthesis of ACh needed to support cholinergic neurotransmission. The choline transporter, designated CHT, was recently cloned. We applied RT/PCR to monitor the expression of CHT in the developing mouse CNS from embryonic day 14 (E14) to postnatal day 30 (P30). We found that CHT was expressed early in development, predominantly in the regions containing cholinergic neurons. In the spinal cord, CHT mRNA was present at close to adult levels at the earliest time point examined (E14) and showed almost no changes after birth. In the striatum and the septum, CHT mRNA increased steadily during embryonic stages and leveled off after birth. Surprisingly, CHT mRNA expression was also detected in other brain regions, notably in the cerebellum, where it peaked on E19, and then rapidly declined during postnatal development. CHT protein was detected by Western blotting as a band of apparent molecular weight of 70 kDa. The accumulation of this protein during development lagged behind mRNA accumulation in all tissues. We also examined the effects of NGF and BMP-4, the potent inducers of choline acetyltransferase and vesicular acetylcholine transporter genes, on CHT expression. Both factors increased CHT mRNA accumulation in primary septal cultures. The effect of NGF was dependent on the PI3K signaling, as it was abolished by the PI3K inhibitor LY294002. This result indicates that some of the signals regulating other cholinergic-specific genes also control CHT expression.

Demethylating agent 5-aza-2'-deoxycytidine enhances expression of TNFRI and promotes TNF-mediated apoptosis in vitro and in vivo.

Promoter hypermethylation within CpG islands plays an important role in the silencing of numerous genes involved in tumor growth including tumor suppressor genes and genes encoding proteins involved in the execution of apoptosis. Here we show that CpG islands are also found within the promoter regions of both human and mouse TNFR1 (TNFRSF1) genes. Selective inhibition of methyltransferases with 5-aza-2'-deoxycytidine increases the expression of TNFR1 in human (WM35) and murine (B16F10) melanoma cells and sensitizes them to TNF-induced apoptosis both in vitro and in vivo. Treatment of mice with the combination of 5-aza-2'-deoxycytidine and recombinant TNF leads to potentiated antitumor effects. Importantly the antitumor efficacy of the combination treatment is shown when both drugs are used in doses that do not exert any antitumor effects when used alone. Altogether our studies show that the combination treatment with 5-aza-2'-deoxycytidine and TNF might be effective in the treatment of melanoma.

Mouse development and cell proliferation in the absence of D-cyclins.

D-type cyclins (cyclins D1, D2, and D3) are regarded as essential links between cell environment and the core cell cycle machinery. We tested the requirement for D-cyclins in mouse development and in proliferation by generating mice lacking all D-cyclins. We found that these cyclin D1(-/-)D2(-/-)D3(-/-) mice develop until mid/late gestation and die due to heart abnormalities combined with a severe anemia. Our analyses revealed that the D-cyclins are critically required for the expansion of hematopoietic stem cells. In contrast, cyclin D-deficient fibroblasts proliferate nearly normally but show increased requirement for mitogenic stimulation in cell cycle re-entry. We found that the proliferation of cyclin D1(-/-)D2(-/-)D3(-/-) cells is resistant to the inhibition by p16(INK4a), but it critically depends on CDK2. Lastly, we found that cells lacking D-cyclins display reduced susceptibility to the oncogenic transformation. Our results reveal the presence of alternative mechanisms that allow cell cycle progression in a cyclin D-independent fashion.

Cerivastatin demonstrates enhanced antitumor activity against human breast cancer cell lines when used in combination with doxorubicin or cisplatin.

Competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase are commonly used in the clinic to treat hypercholesterolemia and have been reported to exert antitumor effects. Cerivastatin is a novel, synthetic and the most pharmacologically potent inhibitor of HMG-CoA reductase. We decided to examine the cytostatic/cytotoxic activity of cerivastatin against human breast cancer cell lines and to test whether the effects of cerivastatin could be potentiated by doxorubicin and cisplatin. Cytostatic/cytotoxic effects of cerivastatin used alone or in the combination with chemotherapeutics were measured with MTT assay. The cell cycle distribution and apoptosis induction were evaluated with flow cytometer. The expression of p21 and p27 cyclin-dependent kinase inhibitors was measured with Western blotting. Isobologram analysis was performed to study the drug interactions. We observed that cerivastatin exerts cytostatic/cytotoxic effects against four human tumor cell lines (T-47D, T4-2, MDA-MB-231, MCF-7). We also demonstrated that cerivastatin exerts growth inhibitory effect through induction of p21 cyclin-dependent kinase inhibitor and inhibition of cell cycle progression. In the two tumor cell lines studied, one sensitive (MDA-MB-231) and one moderately resistant (T4-2) to the cytostatic/cytotoxic effects of cerivastatin we examined the effects of combined treatment with cerivastatin and either doxorubicin or cisplatin. Cerivastatin potentiated cytostatic/cytotoxic effects of cisplatin against T4-2 cells and those of doxorubicin against both cell lines. In T4-2 cells the interaction between doxorubicin and cerivastatin and between cisplatin and cerivastatin was found to be synergistic. Altogether, these studies indicate that cerivastatin is another HMG-CoA reductase inhibitor with potent antitumor effects.

Complete tumour regressions induced by vaccination with IL-12 gene-transduced tumour cells in combination with IL-15 in a melanoma model in mice.

In the present study, IL-12 gene-transduced B78-H1 melanoma cells (B78/IL-12) were used in combination with IL-15 to treat melanoma-bearing mice. Genetically modified B78/IL-12 cells, when injected subcutaneously, induced strong activation of antitumour mechanisms resulting in complete loss of tumourigenicity. In a therapeutic model, intratumoural injection of irradiated B78/IL-12 cells significantly delayed tumour growth and led to the regression of melanoma in one case. Similarly, consecutive daily injections of IL-15 markedly inhibited tumour progression with occasional curative effects. When used in combination, vaccination with B78/IL-12 cells and treatment with IL-15 caused eradication of established tumours in all treated mice. The combined treatment with B78/IL-12 cells and IL-15 activated not only a local response against tumour, but also induced systemic antitumour immunity that led to a delay or inhibition of tumour development at a distant site. In vitro studies demonstrated that when used together, B78/IL-12 cells and IL-15 induced a shift from a type Th2 to a type Th1 response. Activation of the antitumour immune response in double-treated mice resulted, in part, from stimulation of IFN-gamma production and was accompanied by the development of cytotoxic effectors in the spleen. As shown in a macrophage tumouricidal assay, macrophages could also play a role in the antitumour effects. The results confirmed that vaccination with IL-12 gene-modified tumour cells is superior to the treatment with unmodified tumour cell vaccine and, additionally, showed that IL-15 is an excellent candidate for adjuvant therapy, inducing synergistically not only a delay of tumour growth but also its complete eradication.

Interleukin 12-based immunotherapy improves the antitumor effectiveness of a low-dose 5-Aza-2'-deoxycitidine treatment in L1210 leukemia and B16F10 melanoma models in mice.

PURPOSE: Recent findings indicating that many genes related to cancer development are silenced by an aberrant DNA methylation suggest that inhibitors of this process may be effective cancer therapeutics. In this study we investigated the efficacy of low-dose 5-aza-2'-deoxycitydine (DAC), a methylation inhibitor, with interleukin (IL) 12, one of the most potent cytokines with antitumor activity. EXPERIMENTAL DESIGN: Mice inoculated with L1210 leukemia cells or with B16F10 melanoma cells were treated with 7 daily injections of low-dose DAC (0.2 mg/kg) and/or 7 daily doses of IL-12 (100 ng/dose). Scid/scid mice as well as monoclonal antibodies against CD4, CD8, and NK1.1 were used to investigate the mechanisms of the antitumor effects of the combination treatment. The activity of murine lymphocytes was measured with enzyme-linked immunospot and (51)Cr release assays. RESULTS: Treatment with DAC or IL-12 given alone produced moderate antitumor effects. In both tumor models combined treatment resulted in potentiated antitumor effects and produced 70% long-term survivors among mice inoculated with L1210 cells. The antitumor efficacy of combined treatment was abrogated in scid/scid mice, and after depletion of CD4(+) and CD8(+) T cells. Mice inoculated with B16F10 melanoma cells had significantly delayed tumor growth after combined treatment with DAC and IL-12. Strong antitumor effect correlated with a significant activation of lymph node-derived CD8(+) and CD4(+) cells. Transient neutropenia was observed in mice under treatment of DAC alone, but remarkably this effect was not potentiated by IL-12. CONCLUSIONS: This study provides the first evidence that antitumor effects of DAC can be strongly potentiated by IL-12 and could be beneficial in an effective low-dose-based antitumor therapy.

Antitumor effects of photodynamic therapy are potentiated by 2-methoxyestradiol. A superoxide dismutase inhibitor.

Photodynamic therapy (PDT), a promising therapeutic modality for the management of solid tumors, is a two-phase treatment consisting of a photosensitizer and visible light. Increasing evidence indicates that tumor cells in regions exposed to sublethal doses of PDT can respond by rescue responses that lead to insufficient cell death. We decided to examine the role of superoxide dismutases (SODs) in the effectiveness of PDT and to investigate whether 2-methoxyestradiol (2-MeOE(2)), an inhibitor of SODs, is capable of potentiating the antitumor effects of this treatment regimen. In the initial experiment we observed that PDT induced the expression of MnSOD but not Cu,Zn-SOD in cancer cells. Pretreatment of cancer cells with a cell-permeable SOD mimetic, Mn(II)-tetrakis(4-benzoic acid)porphyrin chloride, and transient transfection with the MnSOD gene resulted in a decreased effectiveness of PDT. Inhibition of SOD activity in tumor cells by preincubation with 2-MeOE(2) produced synergistic antitumor effects when combined with PDT in 3 murine and 5 human tumor cell lines. The combination treatment was also effective in vivo producing retardation of the tumor growth and prolongation of the survival of tumor-bearing mice. We conclude that inhibition of MnSOD activity by 2-MeOE(2) is an effective treatment modality capable of potentiating the antitumor effectiveness of PDT.

Erythropoietin restores the antitumor effectiveness of photodynamic therapy in mice with chemotherapy-induced anemia.

PURPOSE: The study was designed to examine the impact of anemia on the antitumor efficacy of photodynamic therapy (PDT) in a murine colon-26 adenocarcinoma model syngeneic with BALB/c mice. EXPERIMENTAL DESIGN: Acute hemolytic anemia was induced by a single i.p. injection of phenylhydrazine hydrochloride (150 mg/kg). Anemia induced by i.p. administration of carboplatin (100 mg/kg) was corrected by s.c. treatment with recombinant human erythropoietin (1000 units/kg/day). The effectiveness of PDT (10 mg/kg Photofrin, 150 J/cm2 laser dose) was evaluated by measurements of the footpad edema and tumor volume. All of the RBC-related parameters were measured from the tail vein. RESULTS: Phenylhydrazine hydrochloride injection resulted in a blunted response of normal tissues to Photofrin-mediated PDT-induced edema formation. Similarly, the antitumor response in mice with hemolytic anemia was nearly completely abrogated. The antitumor effectiveness of PDT was also significantly diminished in a more realistic clinical situation when anemia was induced by administration of carboplatin. Importantly, administration of recombinant human erythropoietin completely restored the sensitivity of the tumor to PDT in carboplatin-treated mice. CONCLUSIONS: These results indicate that anemia can negatively influence the therapeutic effectiveness of PDT. For optimal antitumor response anemia should be corrected before PDT procedure.

IL-12 or IL-15, unlike IL-2, does not interact with histamine in augmenting cytotoxicity of splenocytes against melanoma cells and YAC-1 cells.

It has been suggested that histamine by its ability to downregulate the production of macrophage-derived reactive oxygen species might effectively potentiate the cytotoxic activity of cytokine-stimulated NK cells. Histamine thus reverses negative regulation of NK cells treated with IL-2 and IFN-alpha in the presence of macrophages. We confirm that histamine potently enhances cytotoxic activity of IL-2-stimulated NK cell-enriched splenocytes admixed with macrophages against B16F10 melanoma cells and YAC-1 cells. This stimulation results in production of high amounts of INF-gamma and TNF-alpha. Interestingly, IL-15 by itself promotes production of reactive oxygen species. Although histamine decreased reactive oxygen species production from the cultures of IL-15-stimulated NK cell-enriched splenocytes admixed with macrophages, it did not potentiate the cytotoxicity of IL-15. Further, we demonstrate that histamine-mediated potentiation of cytotoxicity is not applicable to IL-12, another potent activator of NK cell activity.