Transcriptome characterization of male accessory glands in ants to identify molecules involved in their reproductive success.

In insects, seminal fluid proteins that are produced by male accessory glands and transferred to females during mating have key functions in sperm competition and sperm physiology that lead to male reproductive success. In ants, male reproductive success also depends on the longevity of sperm stored in the queen's spermatheca because their sexual offspring are usually produced only after a prolonged storage period. We identified genes that were up-regulated in the male accessory glands relative to the bodies of Crematogaster osakensis to characterize the reproductive molecules associated with male reproductive success in ants. We found novel genes that had no hits in a homology search and that were predominantly expressed in the accessory glands. These reproductive proteins may have evolved under rapid positive selection for reproductive success in the species. Furthermore, we discovered that three spermatheca-specific genes of C. osakensis queens were also enriched in the accessory glands relative to the bodies of males. These genes may be important for maintaining the sperm quality continuously from ejaculation by males to prolonged storage by queens. This research provides crucial information about the molecular mechanisms of sperm maintenance and sexual selection in ants, and also insight into the evolution of reproductive strategies in insects.

Comparison between S+L- assay and LacZ marker rescue assay for detecting replication-competent gammaretroviruses.

To avoid contamination of adventitious gammaretroviruses in biological products such as vaccines, it is necessary to check the master seed cells for manufacturing. There are several assays to detect infectious gammaretroviruses. Among these, sarcoma-positive, leukemia-negative (S+L-) assay is a classical infectivity assay, which is often recommended in governmental guidelines. The S+L- cells used in S+L- assay generate unique focus upon the infection of replication-competent gammaretroviruses. Although S+L- assay is well recognized for the detection, their applicability is questionable in some cases. On the other hand, LacZ marker rescue (LMR) assay detects infectious gammaretroviruses by transducing LacZ marker gene to the target cells, which shows lacZ-positive foci if the infectious virus is present. In this study, we compared LMR and S+L- assays for detection of a variety of endogenous and exogenous gammaretroviruses. As results, LMR assay could detect all gammaretroviruses examined. On the other hand, S+L- assay using feline S+L- cells, termed QN10S, could not detect porcine endogenous retrovirus (PERV) subgroups A/B. Further, S+L- mink cells could not detect feline leukemia virus subgroups B in addition to PERV-A/B. These data indicate that LMR assay is better suited to detect wider range of gammaretroviruses.

Skew in T cell receptor usage with polyclonal expansion in lesions of oral lichen planus without hepatitis C virus infection.

Oral lichen planus (OLP) is a refractory disorder of the oral mucosa. Its predominant symptoms are pain and haphalgesia that impair the quality of life of patients. OLP develops via a T cell-mediated immune process. Here, we examined the characteristics of the infiltrating T cells in terms of the T cell receptor (TCR) repertoires, T cell clonality, T cell phenotypes and cytokine production profiles. TCR repertoire analyses and CDR3 size spectratyping were performed using peripheral blood mononuclear cells (PBMCs) and tissue specimens of OLP biopsies from 12 patients. The cytokine expression profiles and T cell phenotypes were measured by real-time quantitative polymerase chain reaction. We observed that there were skewed TCR repertoires in the tissue samples (TCRVA8-1, VA22-1, VB2-1, VB3-1 and VB5-1) and PBMCs (TCRVA8-1, VB2-1, VB3-1 and VB5-1) from OLP patients. Furthermore, the CDR3 distributions in the skewed TCR subfamilies exhibited polyclonal patterns. We observed increases in CD4(+) T lymphocytes, interleukin (IL)-5, tumour necrosis factor (TNF)-alpha and human leucocyte antigen D-related in the OLP tissue specimens. Taken together, the present results suggest that T cells bearing these TCRs are involved in the pathogenesis of OLP, and that IL-5 and TNF-alpha may participate in its inflammatory process.

Mitochondrial DNA determines androgen dependence in prostate cancer cell lines.

Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic large-deletion mitochondrial DNA is very common in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstrated that the androgen-independent cell line C4-2, established by inoculation of the androgen-dependent LNCaP cell line into castrated mice, has a greatly reduced amount of normal mitochondrial DNA and an accumulation of large-deletion DNA. Strikingly, the depletion of mitochondrial DNA from androgen-dependent LNCaP resulted in a loss of androgen dependence. Reconstitution of normal mitochondrial DNA to the mitochondrial DNA-depleted clone restored androgen dependence. These results indicate that mitochondrial DNA determines androgen dependence of prostate cancer cell lines. Further, mitochondrial DNA-deficient cells formed tumors in castrated athymic mice, whereas LNCaP did not. The accumulation of large deletion and depletion of mitochondrial DNA may thus play a role in the development of androgen independence, leading to progression of prostate cancers.

Osteocalcin promoter-based toxic gene therapy for the treatment of osteosarcoma in experimental models.

Osteocalcin (OC), a noncollagenous bone matrix protein, is expressed in high levels by osteoblasts. To determine whether the OC promoter mediates cell-specific gene expression in cells of osteoblast lineage, we constructed a recombinant adenovirus, Ad-OC-TK, which contains the OC promoter that drives the expression of herpes simplex virus thymidine kinase (TK). We tested the expression of TK by this virus in osteoblast cell lines as well as in non-osteoblastic cell lines by assessing the enzyme activity of TK in vitro. Whereas the OC promoter failed to drive the expression of the TK gene in several non-osteoblastic cell lines such as WH, a human bladder transitional carcinoma, and NIH 3T3, an embryonic mouse fibroblast cell line, the OC promoter mediated high levels of expression in osteoblast cell lines including murine ROS and human MG-63 cells. The addition of acyclovir (ACV), a pro-drug for the inhibition of cell proliferation, resulted in the induction of osteoblast-specific cell death in vitro. Intratumoral injection of Ad-OC-TK into murine ROS osteosarcoma abolished tumor growth in a host treated with subsequent i.p. ACV injection in vivo. The Ad-OC-TK virus plus ACV treatment appears to be highly selective in blocking the growth of both murine and human osteosarcoma cell lines in vitro and murine osteosarcoma in vivo.

Inflammatory gene signature in ulcerative colitis with cDNA macroarray analysis.

BACKGROUND: Most array analyses of ulcerative colitis have focused on identifying susceptibility genes for ulcerative colitis. AIM: To clarify the changes in gene expression during inflammation in ulcerative colitis colon mucosa using cDNA macroarray. METHODS: From 23 ulcerative colitis patients, 16 each of inflamed and non-inflamed specimens (total 32 samples for individual analysis) were obtained by colonoscopic biopsy. Eighteen of the 32 samples, used for pairwise analysis, consisted of nine sample pairs, each pair being from the same patient. We examined expression profiles of approximately 1300 genes with cDNA macroarray. Comparisons were made using two kinds of statistics, t-test and significance analysis of microarray in both analyses. The reproducibility of significant genes from the macroarray analysis was confirmed by real-time ploymerase chain reaction. RESULTS: We detected five upregulated genes, categorized into proinflammatory genes (MRP14, GRO gamma and SAA1) and anti-inflammatory genes (TIMP1 and Elafin) in inflamed mucosa, and one upregulated gene (L-FABP) in non-inflamed mucosa. CONCLUSIONS: As the cDNA macroarray analysis in this study exactly reflects the total profile of gene expression in the clinical setting of ulcerative colitis, the genes identified will be directly applicable to diagnostics or as novel therapeutic targets in active ulcerative colitis.

Potential molecules implicated in downstream signaling pathways of p185BCR-ABL in Ph+ ALL involve GTPase-activating protein, phospholipase C-gamma 1, and phosphatidylinositol 3'-kinase.

Constitutive activation of BCR-ABL tyrosine kinase fusion protein has been shown to be an essential step in the pathogenesis of Philadelphia chromosome (Ph)-positive leukemias. We studied the tyrosine phosphorylated proteins which might be involved in the signaling pathway p185BCR-ABL using a Ph-positive acute lymphoblastic leukemia cell line. p185BCR-ABL but not p145c-abl was constitutively phosphorylated on tyrosine in this cell line. p21ras GTPase-activating protein (GAP) was physically associated with p185BCR-ABL, but not with p145c-abl, and GAP-associated proteins p62/p190 were found to be tyrosine-phosphorylated. Furthermore, p185BCR-ABL was also physically associated with phospholipase C-gamma 1 (PLC-gamma) and phosphatidylinositol 3'-kinase (P13-kinase). Concomitantly, both PLC-gamma and p85 subunit of P13-kinase are tyrosine-phosphorylated in the cells with p185BCR-ABL. These data suggest that GAP, GAP-associated proteins, PLC-gamma, and P13-kinase may participate in downstream signaling for p185BCR-ABL tyrosine kinase.

Combination of 22-oxa-1,25-dihydroxyvitamin D(3), a vitamin D(3) derivative, with vitamin K(2) (VK2) synergistically enhances cell differentiation but suppresses VK2-inducing apoptosis in HL-60 cells.

We originally reported that vitamin K(2) (VK2) effectively induces apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. In addition, VK2 was shown to induce differentiation of leukemia cells when the cells were resistant against VK2-inducing apoptosis. A novel synthetic vitamin D(3)derivative, 22-oxa-1,25-dihydroxyvitamin D(3) (OCT: oxacarcitriol) shows a more potent differentiation-inducing ability among myeloid leukemia cells in vitro with much lesser extent of the induction of hypercalcemia in vivo as compared to the effects of 1alpha,25(OH)(2)D(3). In the present study, we focused on the effects of a combination of OCT plus VK2 on leukemia cells. Treatment of HL-60 cells with OCT for 72 h induces monocytic differentiation. A combination of OCT plus VK2 dramatically enhances monocytic differentiation as assessed by morphologic features, positivity for non-specific esterase staining, and cell surface antigen expressions. This combined effect far exceeds the maximum differentiation induction ability at the optimal concentrations of either OCT or VK2 alone. In addition, pronounced accumulation of the cells in the G0/G1 phase is observed by combined treatment with OCT plus VK2 as compared with each vitamin alone. In contrast to cell differentiation, caspase-3 activation and apoptosis induction in response to VK2 are significantly suppressed in the presence of OCT in HL-60 cells. These data suggest that monocytic differentiation and apoptosis induction of HL-60 cells are inversely regulated. Furthermore, pronounced induction of differentiation by combined treatment with VK2 plus OCT was also observed in four out of six cases of primary cultured acute myeloid leukemia cells in vitro, suggesting that VK2 plus OCT might be a potent combination for the differentiation-based therapy for acute myeloid leukemias.

Apoptosis/differentiation-inducing effects of vitamin K2 on HL-60 cells: dichotomous nature of vitamin K2 in leukemia cells.

We originally reported that vitamin K2 (VK2) analogs, including menaquinone 4 (MK4) but not vitamin K1, effectively induce apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. It has also been reported by others that VK2 showed the differentiation-inducing activity in leukemia cell lines. To investigate the discrepancy between apoptosis- and differentiation-inductions of leukemia cells by VK2 treatment, we used bcl-2 gene transfected HL-60 cells (HL-60-bcl-2) which resulted in five-fold over-expression of BCL-2 protein, and then compared the effects of MK4 to the control HL-60-neo cells. Seventy-two hours of exposure to various concentrations of MK4 resulted in growth inhibition of these cells in a dose-dependent manner (0.1-50 microM), however, HL-60-bcl-2 was less sensitive against MK4. MK4 potently induced apoptosis of HL-60-neo cells along with the depolarization of mitochondrial membrane potential and caspase-3 activation. Notably, HL-60-bcl-2 was almost completely resistant to apoptosis induction in response to MK4, although cell growth inhibition was still observed. In spite of the abrogation of apoptosis induction, about 90% of HL-60-bcl-2 cells were arrested in the G0/G1 phase within 48 h of exposure to 10 microM of MK4 accompanied by up-modulation of p27KIP1 expression. Concomitantly, HL-60-bcl-2 cells underwent monocytic differentiation. These data suggest that VK2 also shows the differentiation inducing effects on leukemia cells which are resistant against VK2-inducing apoptosis. The dichotomous nature of VK2 against leukemia cells appears to have clinical benefits for the treatment of patients with leukemias and myelodysplastic syndromes.

Vitamin K2 selectively induces apoptosis of blastic cells in myelodysplastic syndrome: flow cytometric detection of apoptotic cells using APO2.7 monoclonal antibody.

We have previously reported that vitamin K2 (VK2) but not VK1 has a potent apoptosis-inducing effect on freshly isolated leukemia cells from patients with various types of leukemia. By multi-color flow cytometric analysis using monoclonal antibody (mAb), APO2.7, which detects mitochondrial 7A6 antigen specifically expressed by cells undergoing apoptosis, we further investigated the apoptosis-inducing effect of VK2 on minor populations of leukemic blast cells in bone marrow from patients with myelodysplastic syndrome (MDS) and overt myeloid leukemia (post-MDS AML). Limiting dilution of CD95 (anti-Fas) mAb-treated apoptotic Jurkat cells with nonapoptotic CTB-1 cells revealed that APO2.7-positive Jurkat cells were consistently detectable by flow cytometry when present at levels of at least 5% in the CTB-1 suspension. In patient samples the gating area for leukemic clone was determined using cell surface antigen-specific mAbs conjugated with either fluorescein isothionate (FITC) or phycoerythrin (PE) and subsequently the cells stained with phycoerythrin cyanine (PE-Cy5)-conjugated APO2.7 mAb were assessed within the gating area of the leukemic clone for monitoring apoptosis. Treatment of the bone marrow mononuclear cells with 3-10 microM of VK2 (menaquinone-3, -4 and -5) in vitro potently induced apoptosis of the leukemic blast cells as compared with the untreated control cells in all 15 MDS patients tested. This effect was more prominent on blastic cells than that on mature myeloid cells such as CD34-/CD33+ gated cells. In addition, VK2 performed much less effectively on CD3-positive lymphoid cells. In contrast to VK2, VK1 did not show apoptosis-inducing activity. These data suggest that VK2 may be used for treatment of patients with MDS in blastic transformation.

Tyrosine phosphorylation and activation of focal adhesion kinase (p125FAK) by BCR-ABL oncoprotein.

Focal adhesion kinase (p125FAK; FAK) is a protein tyrosine kinase that is tyrosine-phosphorylated in response to v-src-mediated transformation, cell adhesion, and stimulation with neuropeptides. To elucidate a possible functional relationship between FAK and BCR-ABL oncoprotein detected in Philadelphia chromosome-positive (Ph+) leukemias, we investigated the tyrosine phosphorylation state of FAK in a murine growth factor-dependent cell line and in its stable human bcr-abl cDNA transfectant. In interleukin-3 (IL-3)-dependent NFS/N1.H7 cells, tyrosine phosphorylation of FAK was not detected after stimulation with either IL-3 or Steel factor (SLF), both of which involve Ras-mediated signaling pathways. However, stable gene transfection with p210bcr-abl cDNA into H7 cells made these cells growth factor-independent for proliferation and resulted in constitutive tyrosine phosphorylation and kinase activation of FAK. Constitutive phosphorylation and activation of FAK was also observed in all Ph+ leukemia cell lines examined--that is, K562, TS9;22, and YS9;22, which express p210BCR-ABL, and NALM-21 and OM9;22, which express p185BCR-ABL. Ph-negative (Ph-) cell lines, such as MO7e and JM, did not show any detectable tyrosine phosphorylation of FAK. FAK phosphorylation in BCR-ABL-expressing cells was inhibited in a dose-dependent manner by cytochalasin D, a reagent that disrupts the intracellular network of actin filaments. However, no suppression of kinase activity or protein expression of BCR-ABL was observed after treatment with cytochalasin D. A physical association between BCR-ABL and FAK was not apparent. These data suggest that BCR-ABL may be involved in the activation of FAK. Moreover, FAK may be distinct from components in Ras-mediated signaling cascades that are activated by stimulation of myeloid cells with various cytokines.

Molecular therapy with recombinant p53 adenovirus in an androgen-independent, metastatic human prostate cancer model.

The lethal phenotypes of advanced prostate cancer are androgen independent (AI) and metastatic to the axial skeleton. Our laboratory has developed an AI mouse model of metastatic human prostate cancer. In this communication, we report the development of tumor suppressor gene therapy in this AI and metastatic (C4-2) cancer model. By using recombinant adenovirus as a delivery vehicle, we introduced a wild-type p53 tumor suppressor gene into prostate cancer cell lines. Despite a silent mutation at codon 152 of the p53 gene, C4-2 cells express functional, but low, levels of p53 protein. However, the other prostatic cell lines, PC-3 and DU145, have a deletion mutation and two point mutations of the p53 gene, respectively. In vitro studies showed that cell growth, as measured by the thymidine incorporation assay, was inhibited in the C4-2, PC-3, and DU145 cells infected with wild-type p53 adenovirus in comparison to control viruses. Recombinant wild-type p53 adenovirus inhibited prostate tumor growth and its production of prostate-specific antigen (PSA) when injected into C4-2 tumors in nude mice. All p53-treated mice were tumor free as long as 12 weeks after cessation of the 8-week treatment regimen. Two of 8 p53-treated mice developed small tumors growing at distant sites after a prolonged period of follow-up observation. Moreover, other AI prostate cancer cells, PC-3 and DU145, treated with Ad5-CMV-p53 failed to develop into tumors in vivo. This gene therapy strategy may be used against AI prostatic cancer regardless of p53 gene mutation status.

Pharmacokinetics of natural human IFN-alpha in hemodialysis patients.

A pharmacokinetic study of natural human interferon-alpha (IFN-alpha) was conducted in hemodialysis patients. Natural human IFN-alpha was intramuscularly (i.m.) administered to 8 hemodialysis patients at a single dose of 5 million IU and to 7 patients undergoing hemodialysis at the same dose once daily for 5 successive days. The serum antiviral activity was determined by a cytopathic effect bioassay. In the single dose study, the serum antiviral activity reached a maximum (Cmax) of 56.4 +/- 33.3 IU/ml at 8.3 +/- 2.7 h after dosing, and the area under the serum concentration-time curve (AUC0-24h) was 957.2 +/- 601.8 IU h/ml. The Cmax and AUC0-24h values at day 5 following the repeated dosing were both 2.6-fold higher than those of day 1, and the serum antiviral activity reached a steady state within 3 days after initiation of repeated administration. The serum antiviral activity in hemodialysis patients showed a tendency to increase compared with that in the subjects with normal renal function, but the magnitude of the differences was not great. In one nonhemodialysis patient with poor renal function (creatinine clearance < 30 ml/min), no increases in serum antiviral activity owing to repeated dosing were observed. The main adverse events seen were fever (4 of 13, 30.8%), leukopenia (3 of 13, 23.1%), and fatigue (2 of 13, 15.4%). These results suggest that dosage modifications of natural human IFN-alpha are unnecessary for patients with low renal function, even those undergoing hemodialysis.

Chemogene therapy: osteocalcin promoter-based suicide gene therapy in combination with methotrexate in a murine osteosarcoma model.

We previously reported that the recombinant adenovirus (Ad) vector containing the thymidine kinase (TK) gene driven by the osteocalcin (OC) promoter (Ad-OC-TK), when delivered concurrently with acyclovir (ACV), is highly selective in blocking the growth of osteosarcoma in experimental models (Cancer Res. 1996;56:4614-4619). To investigate the possible additive effects of the combined treatment of gene therapy and conventional chemotherapy (chemogene therapy), we compared the effect of low dose (IC10) methotrexate (MTX) and OC promoter-based toxic gene therapy with either of these single modalities alone. We choose low dose MTX with the intent of determining whether chemosensitization of the osteosarcoma may be possible in combination with gene therapy with an overall reduced toxicity profile and enhanced therapeutic efficacy when compared to a single agent alone. In vitro, the combined treatments of MTX (3 ng/mL) and Ad-OC-TK (20 multiplicity of infection (MOI)/target cell) plus ACV (10 mg/mL) had an additive therapeutic effect over that of either MTX (P < 0.05) or Ad-OC-TK plus ACV treatment alone (P < 0.05). In vivo, nude mice with subcutaneous tumors of either human osteosarcoma (MG-63) or rat osteosarcoma (ROS) received three intratumoral injections of Ad-OC-TK (5 x 10(8) PFU) plus daily intraperitoneal ACV (40 mg/kg body weight) for 2 weeks in combination with five weekly bolus intraperitoneal MTX (87.5 mg/kg). Osteosarcoma tumor growth was inhibited more efficiently than by either Ad-OC-TK plus ACV (P < 0.05) or MTX treatment (P < 0.005) alone. At day 45 in the ROS group, 100% of the animals survived when treated with chemogene therapy, whereas 80% survived with gene therapy and no animals survived in the MTX-treated or untreated controls. In summary, we developed a novel therapeutic strategy for the treatment of osteosarcoma employing both chemotherapy and gene therapy. Chemogene therapy could potentially achieve better antitumor effects with reduced toxicity than the conventional chemotherapy or gene therapy protocols alone.

In vivo suppression of osteosarcoma pulmonary metastasis with intravenous osteocalcin promoter-based toxic gene therapy.

Pulmonary metastases are the main cause of death of patients with several types of cancer, including osteosarcoma, renal cell carcinoma, malignant melanoma, and breast cancer. Previously, we demonstrated that intralesional injection of the recombinant adenovirus (Ad) vector containing the herpes simplex virus thymidine kinase (TK) gene driven by an osteocalcin (OC) promoter (Ad-OC-TK) effectively suppressed the growth of osteosarcoma cells in vitro and tumors in vivo in a tumor-specific manner when supplemented with the prodrug acyclovir (ACV). In this communication, we studied the potential efficacy of the treatment of osteosarcoma pulmonary metastases with a systemic delivery route of Ad-OC-TK supplemented with ACV. We established osteosarcoma lung metastases in nude mice by the intravenous injection of rat osteosarcoma cells, ROS 17/2.8. These cells colonized and formed tumor nodules within 1 week in the lungs of nude mice. Whereas systemic delivery of a recombinant Ad vector containing the Escherichia coli beta-galactosidase (beta-gal) gene driven by a Rous sarcoma virus universal promoter (Ad-RSV-beta-gal) resulted in the nonspecific expression of beta-gal activity in the lung parenchyma, Ad-OC-beta-gal administration resulted in specific beta-gal expression in tumor cells deposited in the lung. When nude mice bearing ROS 17/2.8 lung tumors were treated with systemic Ad-OC-TK through tail vein administration, subsequent intraperitoneal ACV treatment significantly decreased the number of tumor nodules (P < .0001) and the net lung wet weight (P = .0005) while significantly increasing (.005 < P < .01) the survival of animals, when compared with untreated and Ad-OC-TK- or ACV-treated control groups. These results suggest that Ad-OC-TK/ACV may be used as a systemic therapy for the treatment of osteosarcoma lung metastasis.

Additional gene therapy with Ad5CMV-p53 enhanced the efficacy of radiotherapy in human prostate cancer cells.

PURPOSE: The aim of this study was to investigate the efficacy of combination therapy of ionizing radiation (IR) and adenoviral p53 gene therapy and to evaluate its molecular mechanisms. METHODS AND MATERIALS: Two human prostate cancer cell lines, DU145 and PC-3 cells, containing different types of p53 gene mutations, were investigated. The recombinant adenovirus vector containing the wild-type p53 gene (Ad5CMV-p53) was used for this study. Cells were irradiated (in 0, 2, 4, and 6 Gy, 300 cGy/min) and after 12 h of irradiation, the cells were infected with various doses of Ad5CMV-p53 (0-40 multiplicity of infection [MOI]). Cytotoxicity was determined by clonogenic assay. The molecular mechanisms were evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), apoptotic cell detection, and cell cycle analysis. RESULTS: The cell growth inhibition in DU145 (p53-mutated) cells by IR was strongly enhanced by additional Ad5CMV-p53 infection in a viral dose-dependent manner. In DU145 cells, IR alone induced minimal p53 mRNA expression. However, IR combined with Ad5CMV-p53 infection stimulated significant increase in p53 mRNA expression supplemented with Bax and p21 mRNA expressions. In PC-3 (p53-null), IR induced Bax and p21 mRNA expression, while the combination effects were observed in p53, Bax, and p21 mRNA expression. Apoptotic cell deaths were rarely observed after IR alone (DU145: 3%, PC-3: 5%). However, after combination therapy, the proportion of apoptotic cells greatly increased (sevenfold in DU145 cells, and twice in PC-3 cells). G1 cell cycle arrest was observed after Ad5CMV-p53 infection and the combination in both cell lines. CONCLUSION: In this study, we demonstrated that the combination of IR and Ad5CMV-p53 gene therapy resulted in remarkable synergistic effects in human prostate cancer cells. This combination therapy could be one of the optimal treatment strategies for radioresistant prostate cancer.

Inhibitory effect of a nucleoside analog, acyclovir, on leukemia cells.

Acyclovir (ACV), a nucleoside analog, has been demonstrated previously to suppress selectively the proliferation of NIH3T3 fibroblastic cells transformed by either v-abl or bcr-abl gene transfection. From a viewpoint of clinical application of ACV, we investigated whether ACV inhibited the growth of leukemia cells expressing either p210 BCR-ABL or p185BCR-ABL. Acyclovir exerted an inhibitory effect on OM9;22 cells, p185BCR-ABL expressing cells, in a dose-dependent manner. Despite no down-modulation of a BCR-ABL tyrosine kinase activity or its expression was observed after treatment with ACV, cell cycle analysis demonstrated synchronization of OM9;22 cells at the G0/G1 phase. This suggests that, although ACV does not directly act on BCR-ABL tyrosine kinase, ACV may exert its inhibitory effect on some leukemia cell lines via alterations of the cell cycle. Although selective inhibition of Philadelphia chromosome-positive leukemia cell growth was not apparent, our data provides a therapeutic possibility for ACV in the treatment for leukemia.

Establishment of a novel B-lymphoma cell line, CTB-1, with strong Pas antigen expression having chromosomal translocation (14;22).

We established a new lymphoma cell line, designated CTB-1, from pericardial effusion of a patient with diffuse large B-cell lymphoma. This cell line showing vigorous growth ability has undergone 260 passages over a period of 34 months in suspension culture, and is heterotransplantable to nude mice. The cultured cells were positive for CD10, CD19, CD20, CD21, HLA-DR, and surface IgG kappa, and negative for T cell antigens. Chromosomal analysis revealed a t(14;22)(q32;q11) that is consistent with original lymphoma cells. CTB-1 cells show the high cell surface expression level of Fas antigen/APO-1. However, ligation of Fas antigen with anti-Fas monoclonal antibody (clone CH-11) did not induce apoptosis of CTB-1 cells. This suggests that Fas itself or the downstream signaling pathways of Fas may be impaired in this cell line. This new cell line may provide a useful in vitro system to study the biology and pathogenesis of B-cell lymphoma which is independent of Fas-mediated apoptosis.

Effects of retinoic acid and interferon-gamma on expression of the retinoic acid receptor in mouse renal cell carcinoma.

The clinical outcome of interferon-gamma treatment of metastatic renal cell carcinoma remains unsatisfactory. To overcome this, a reagent that has a different action on cancer cells is desired. One such candidate may be 13-cRA (cis retinoic acid), a vitamin A derivative known to markedly regulate the differentiation and proliferation of normal and neoplastic cells. Moreover, 13-cRA demonstrates a remarkable synergistic effect with IFN-gamma in certain types of cancer. We investigated the efficacy of concomitant administration of 13-cRA and IFN-gamma. The in vivo anti-tumor effects were evaluated in a lung metastasis model using a mouse renal cell carcinoma cell line (RenCa). The in vitro anti-tumor effects were also assessed by MTT assay. The presence of retinoic acid receptors (RAR)-alpha, -beta and -gamma was examined by PCR analysis. The influence of IFN-gamma on these retinoic acid receptors was evaluated by Northern blotting. IFN-gamma showed anti-tumor effects both in vivo and in vitro. This effect was enhanced synergistically with concomitant 13-cRA treatment. RenCa cells expressed RAR-alpha, and -gamma, but not -beta according to PCR analysis. IFN-gamma treatment increased the expression level of RAR-alpha and RAR-gamma according to Northern blot analysis. Combination therapy using IFN-gamma and 13-cRA showed synergistic anti-tumor effects, which exceeded those of each therapy alone. Moreover, IFN-gamma treatment increased RAR-alpha and RAR-gamma expression. Thus, the augmented anti-tumor effects of IFN-gamma and 13-cRA may be attributable to enhanced expression of RAR-alpha and RAR-gamma by IFN-gamma treatment.

A G-quadruplex-interactive agent, telomestatin (SOT-095), induces telomere shortening with apoptosis and enhances chemosensitivity in acute myeloid leukemia.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is up-regulated in the vast majority of human neoplasias but not in normal somatic tissues. Therefore, the telomerase complex represents a promising universal therapeutic target in cancer. Telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We examined G-quadruplex interactive agent, telomestatin (SOT-095), for its ability to inhibit the proliferation of human leukemia cells, including freshly obtained leukemia cells. Telomere length was determined by either the terminal restriction fragment method or flow-FISH, and apoptosis was assessed by flow cytometry. Moreover, chemosensitivity was examined in telomestatin-treated U937 cells before ultimate telomere shortening. Treatment with telomestatin reproducibly inhibited telomerase activity in U937 and NB4 cells followed by telomere shortening. Enhanced chemosensitivity toward daunorubicin and cytosine-arabinoside was observed in telomestatin-treated U937 cells, before ultimate telomere shortening. Telomere shortening associated with apoptosis by telomestatin was evident in some freshly obtained leukemia cells from acute myeloid leukemia patients, regardless of sub-types of AML and post-myelodysplasia AML. These results suggest that disruption of telomere maintenance by telomestatin limits the cellular lifespan of AML cells, as well. However, in a minority of AML patients apoptosis was not evident, thus indicating that resistant mechanism might exist in some freshly obtained AML cells. Therefore, further investigation of telomestatin as a therapeutic agent is warranted.