Synergistic inhibitory effects of interferon-alpha and 5-fluorouracil on meningioma cells in vitro

We have investigated the effects of interferon- (IFN-) and 5-fluorouracil (5-FU) on meningioma cells in two different culture systems, evaluated by the uptake of radiolabelled methionine. With both IFN- and 5-FU an inhibitory effect on the uptake of radiolabelled methionine by the meningioma cells was demonstrated, and we found a synergistic inhibitory effect with a combination of IFN- and 5-FU. To obtain a maximal inhibition of cell metabolism without causing cell toxicity, we were able to decrease the dose of 5-FU by simultaneously adding IFN-. Our results suggest that a combined treatment of IFN- and 5-FU may be a successful alternative for patients with inoperable meningiomas. A novel in vitro positron emission tomography technique was used for the study of metabolic changes in tumour cells caused by drug treatment, which is complementary to conventional cell culture techniques.     

Prediction of BRCA1 and BRCA2 mutation status using post-irradiation assays of lymphoblastoid cell lines is compromised by inter-cell-line phenotypic variability

BACKGROUND AND PURPOSE: Assays to determine the pathogenicity of unclassified sequence variants in disease-associated genes include the analysis of lymphoblastoid cell lines (LCLs). We assessed the ability of several assays of LCLs to distinguish carriers of germline BRCA1 and BRCA2 gene mutations from mutation-negative controls to determine their utility for use in a diagnostic setting. MATERIALS AND METHODS: Post-ionising radiation cell viability and micronucleus formation, and telomere length were assayed in LCLs carrying BRCA1 or BRCA2 mutations, and in unaffected mutation-negative controls. RESULTS: Post-irradiation cell viability and micronucleus induction assays of LCLs from individuals carrying pathogenic BRCA1 mutations, unclassified BRCA1 sequence variants or wildtype BRCA1 sequence showed significant phenotypic heterogeneity within each group. Responses were not consistent with predicted functional consequences of known pathogenic or normal sequences. Telomere length was also highly heterogeneous within groups of LCLs carrying pathogenic BRCA1 or BRCA2 mutations, and normal BRCA1 sequences, and was not predictive of mutation status. CONCLUSION: Given the significant degree of phenotypic heterogeneity of LCLs after gamma-irradiation, and the lack of association with BRCA1 or BRCA2 mutation status, we conclude that the assays evaluated in this study should not be used as a means of differentiating pathogenic and non-pathogenic sequence variants for clinical application. We suggest that a range of normal controls must be included in any functional assays of LCLs to ensure that any observed differences between samples reflect the genotype under investigation rather than generic inter-individual variation.     

Extremely acute exacerbation of interstitial pneumonia after interferon-alpha treatment for metastatic renal cell carcinoma

We report the case of a 62-year-old Japanese man who presented with right renal cell carcinoma and multiple metastases. The patient had a past medical history of idiopathic interstitial pneumonia 6 years previously. He had received pulse corticosteroid therapy and oral prednisolone, which resulted in marked clinical improvement. He had been followed up for the interstitial pneumonia, without medication, for 5 years and the idiopathic interstitial pneumonia was inactive when the metastatic renal cell carcinoma was diagnosed. However, the patient presented with extremely acute exacerbation of the interstitial pneumonia that occurred after only three intramuscular injections of standard-dose interferon-alpha. Urologists should be aware of this complication in the treatment of renal cell carcinoma patients who have a prior history of interstitial pneumonia.     

Desensitization of human breast-cancer cells by interferon-alpha

Continuous exposure of human breast cancer cells, MCF-7, to interferon-alpha (IFN) induces a state.of non-responsiveness termed as desensitization. mRNA 561 is transiently induced by IFN-alpha in MCF-7 cells, peak cytoplasmic levels are reached by six to twelve hours; the mRNA level declines steadily and is reduced to uninduced levels by forty eight hours. Induction of mRNA 561 was used as an index of responsiveness of cells to IFN-alpha and desensitization was characterized in MCF-7 cells and in MCF-7 cells transfected by the v-H-ras oncogene (MCF-7ras). The kinetics and degree of IFN-mediated induction of mRNA 561 was comparable in both the cell lines. Desensitization was observed in MCF-7 cells and not in MCF-7ras. It was a reversible event, requiring de novo protein synthesis as inclusion of cycloheximide inhibited desensitization. The cellular elements that mediate such a phenomena are elicited by IFNs during the initial phases of IFN action and may be polypeptides. The refractory period, the time after which MCF-7 cells become responsive, was determined to be five days. In conclusion, we demonstrate the use of mRNA 561 induction in evaluating desensitization. Inhibition of protein synthesis or transfection with ras blocks desensitization in MCF-7 cells.     

Combination therapy of human pancreatic cancer implanted in nude mice by oral fluoropyrimidine anticancer agent (S-1) with interferon-alpha

Purpose: We evaluated the antitumor and antiangiogenic activities of human natural interferon-alpha (IFN-α) alone or in combination with S-1 against human pancreatic cancer cells. Methods: Three days after the subcutaneous (s.c.) implantation of tumor cells, mice (n = 12) were received s.c. injection with IFN-α alone (10,000 U six times a week), oral administration with S-1 alone (8 mg/kg six times a week), or both with IFN-α and S-1 (8, 10, 12 mg/kg six times a week). Results: Administration of IFN-α in combination with S-1 significantly decreased progressive growth and angiogenesis of human pancreatic cancer cells. The combination therapy produced more significant inhibition in expression of the representative proangiogenic molecules, vascular endothelial growth factor and basic fibroblast growth factor than individual treatment either IFN-α or S-1 alone did. These treatments also decreased the staining of proliferating cell nuclear antigen, induced apoptosis and decreased microvessel density. In order to better understand the precise molecular mechanisms by which IFN-α and S-1 exert its effects, we have utilized cDNA microarray including 124 known genes to determine the gene expression profile altered by IFN-α and S-1 treatment. We found a total of seven genes which showed a twofold change after IFN-α and S-1 treatment in addition to VEGF, bFGF, CD31, MMP-2, MMP-7 and MMP-9. Among these genes, we found down-regulation of six genes and up-regulation of one gene, which are related to angiogenesis, tumor cell invasion and metastasis. Conclusions: These data suggest that administration of IFN-α in combination with S-1 may provide a novel and effective approach to the treatment of human pancreatic cancer.     

Synergistic antitumour effect of a combination of toremifene and interferon-alpha on ZR-75-1 human breast cancer cells: dependence on interferon-alpha subtype

We investigated the effect of toremifene, interferon-alpha2a, interferon-alpha2b and interferon-alpha2c, singly and in combination for their effect on the growth of ZR-75-1 human breast cancer cells. Median effect analysis was used to determine synergistic or additive effects. Anti-proliferative studies showed that the growth of ZR-75-1 cells was inhibited to a greater extent by combination treatment with toremifene plus interferon-alpha2a, resulting in a synergistic interaction (CI <1) for all concentrations tested. A combination of toremifene plus interferon-alpha2b resulted in a synergistic interaction (CI <1) for the two highest concentrations of toremifene (10(-6) and 10(-7) M) and an additive effect (CI approximately equal to 1) for the lower concentrations (10(-8) to 10(-10) M). When toremifene was combined with interferon-alpha2c no additive or synergistic interaction was determined.     

Interferon production and activation of non-specific effector cells by stimulation with lymphoblastoid cell lines in vitro

A population of non-specific effector lymphocytes is generated in response to stimulation with lymphoblastoid cell lines (LCL). These cytotoxic cells have an activity analogous to that exhibited by natural killer (NK) cells. When lymphoid cells lines established by transformation with Epstein-Barr virus (EBV) are used to stimulate autochthonous T-cell-enriched lymphocytes, the activity against the NK-sensitive target, K562, is increased up to 14-fold. The stimulated T lymphocytes produce interferon, and this factor augments the cytotoxic activity of unstimulated cells. The size distribution of cytotoxic lymphocytes after stimulation with the autochthonous EBV line is unlike that of either T-cells or interferon-augmented T-cells. Autochthonous stimulated lymphocytes which kill K562 are of several size classes, and are included in populations containing large blast cells. In contrast, the K562 activity of both unstimulated T-cells and interferon-augmented T-cells is contained in a more discrete population of cells, just slightly larger than the majority of lymphocytes. Thus, the generation of non-specific effector cells during stimulation with lymphoblastoid cell lines appears to involve the activation of a population of blast-size cells in addition to those initially responsive to interferon augmentation.     

Treatment of metastatic renal cell carcinoma with a combination of human lymphoblastoid interferon-alpha and cimetidine

Human lymphoblastoid interferon-alpha was administered intramuscularly at a dose of 5 x 10(6) units/day to 20 metastatic renal cell carcinoma patients. For potentiating the antitumor effect of interferon, cimetidine was also given to them orally at a dose of 800 mg/day. The combination therapy obtained a complete response in three patients (15%) and a partial response in three (15%). Nine patients (45%) had stable disease and five (25%), progressive disease. All six patients who responded to the combination therapy had been nephrectomized and had pulmonary metastases. Two of them also had metastases to other sites (mediastinal lymph nodes and bone). The pulmonary metastases were significantly more receptive to interferon therapy than those at the other sites. The average times before a response was obtained were 2.2 months for a minor response, 2.7 months for a partial response and 3.0 months for a complete response, and the average duration of response was 26 months. The six patients who responded survived for a significantly longer period than the 14 non-responding patients treated with interferon in combination with cimetidine. The major toxicities encountered were fever, fatigue and anorexia due to interferon, and the combination therapy was well tolerated except in three patients. The results suggest that interferon-alpha and cimetidine combination therapy may be of use in the management of patients with metastatic renal cell carcinoma.     

Effect of sidestream and mainstream smoke exposure on in vitro interferon-alpha/beta production by L-929 cells

A peristaltic pump smoking machine that allows simultaneous generation of mainstream (active) and sidestream (passive) smoke from a cigarette was used to expose cultures of murine L-929 cells, a potent producer of interferon, to smoke. The cigarette used was the University of Kentucky 2R1 reference cigarette. The dosages of smoke used for exposure were the highest doses possible that generated a minimum toxic effect, and they were serially diluted to lower doses. Dosages were determined by the number of smoke puffs generated, the volume of smoke puffs generated, and the total particulate matter deposited on Cambridge filters in the smoke machine. Viability of exposed cells was equivalent to control cell cultures. Interferon-alpha/beta was induced by addition of polyriboinosinic-polyribocytidylic acid to the cells. Interferon production was substantially reduced in viable cells exposed to mainstream or sidestream smoke. Aging of smoke by delaying time of exposure of the cells to the smoke, or filtration of smoke through activated charcoal substantially decreased the alteration of interferon production by smoke exposure. These results suggest that actual exposure of cells to mainstream or sidestream smoke can inhibit in vitro interferon-alpha/beta induction, but the cells can be protected from these effects by smoke manipulation.     

Post-translational regulation of COX2 activity by FYN in prostate cancer cells

While increased COX2 expression and prostaglandin levels are elevated in human cancers, the mechanisms of COX2 regulation at the post-translational level are unknown. Initial observation that COX2 forms adduct with non-receptor tyrosine kinase FYN, prompted us to study FYN-mediated post-translational regulation of COX2. We found that FYN increased COX2 activity in prostate cancer cells DU145, independent of changes in COX2 or COX1 protein expression levels. We report that FYN phosphorylates human COX2 on Tyr 446, and while corresponding phospho-mimetic COX2 mutation promotes COX2 activity, the phosphorylation blocking mutation prevents FYN-mediated increase in COX2 activity.     

高转移肝癌MHCC97细胞中JAK/STATs通路相关分子表达的研究

目的：干扰素α能明显抑制裸鼠MHCC97肝癌移植瘤的生长，但对该细胞的增殖无影响。本研究着重探讨导致MHCC97细胞对干扰素α无应答的分子机理。方法：用RT—PCR、免疫印迹及核苷酸测序等方法研究MHCC97细胞中JAK／STATs通路上干扰素α受体、irf9、stαtj及stat2基因的转录和蛋白的表达。结果：MHCC97细胞构成性转录和翻译statl、stat2分子；虽同时转录一定量的irf9 mRNA，但检测不到相应的蛋白；测序分析发现irf9基因的核苷酸组成存在几个点突变。结论：由irf9基因突变所引起的JAK／STATs信号转导功能的不完整，可能是该细胞对干扰素α增殖抑制无应答的分子原因之一。     

Quantitative analysis of cytotoxicity induced by HTLV-I carrying cells against a human lymphoblastoid cell line, Molt-4

Co-cultivation of HTLV-I carrying cells and virus-free lymphoid cells resulted in the cytopathic effect and cytotoxicity of the latter cells. Microscopically, these phenomena were observed as early as 2 h as the appearance of multinuclear giant cells and ballooning cells with striking resemblance with cytopathic changes induced by HTLV-III. When the cytopathic effect and cytotoxicity of the target cells by HTLV-I carrying cells were assayed by 51Cr-release assay system, these phenomena were quantitatively analysed and the cytotoxic activity was observed generally in HTLV-I positive cells. Virally induced cytotoxicity was inhibited by plasma of adult T-cell leukemia (ATL) patients specifically. Cytotoxic activity of HTLV-I positive cell lines was correlated with HTLV-I antigen expression of them. HTLV-I negative cell lines did not express cytotoxicity significantly.     

Endogenous production of IL-8 by human colorectal cancer cells and its regulation by cytokines

This study investigated the endogenous production of the pro-inflammatory and angiogenic cytokine IL-8 by human colorectal cancer cells. We describe substantial (>0.5 ng/ml) constitutive production of IL-8 by certain human colorectal cancer cell lines without the requirement for exogenous stimulation. Tumour cell-derived IL-8 production was upregulated by the addition of the pro-inflammatory type 1 cytokines TNF alpha, IL-1 beta and IFN gamma. Addition of the regulatory type 2 cytokines IL-4 and IL-10 produced paradoxical stimulation of IL-8 production in certain cell lines and downregulation of IL-8 production in others. These results suggest that tumour-derived cytokine production may have an important role in patients with colorectal cancer. Furthermore, they demonstrate the complexity of tumour cell cytokine production and highlight the difficulties in developing effective therapeutic biological response strategies.     

Mechanism of action of interferon-alpha 2 in hairy cells and lymphoblastoid cell lines: the significance of type I interferon receptors and RNA synthesis

Lymphoblastoid cells were subjected to Western Blot analysis for detection of Interferon-alpha-binding proteins. JOK-1 cells--a human hairy cell leukemia line--revealed three proteins with apparent molecular weights of 120, 100 and 32 kD, respectively. Down-regulation of the receptor was observed. A differential binding pattern of two proteins (100 and 85 kD) was observed in T-CLL, whereas no signal detection was achieved in B-CLL. Mononuclear cells from 6 patients with hairy cell leukemia and 6 patients with CLL were found to differ significantly in terms of nucleic acid precursor incorporation.     

Elaboration of pyrimidine-specific nucleosidases by human lymphoblastoid cells of established cultures.

Pyrimidine-specific nucleosidases were released rapidly by human lymphoblastoid cells of established cultures when incubated under certain culture conditions having no adverse affect on their viability or morphology. Nucleosidase production was not restricted to any particular type of lymphoblastoid line; enzymes with a high level of activity were elaborated by cells of cultures initiated from healthy subjects and patients with uncontrolled lymphocytic or myelocytic leukemia, as well as by cells of cultures exhibiting mostly B- or T-cell properties. Tritiated thymine and uracil, which were not incorporated to any appreciable extent by DNA- and RNA-synthesizing cells, were identified by paper chromatography as the primary products arising from nucleosidase degradation of radiolabeled thymidine, uridine, and cytidine. Neither adenosine nor guanosine was catabolized. These heat-labile and ultraviolet-sensitive enzymes with a molecular weight of 5 to 10 X 10(4) did not affect the viability, morphology, or proliferation of lymphocytes in mitogenactivated cultures, lymphoblastoid cells in long-term cultures, or fibroblasts in monolayer cultures.     

IL-6 and IFN-gamma regulation of IL-10 production by human colon carcinoma cells

IL-10 has been shown to play a crucial role in immunosuppression in cancer patients. We explored the regulation of IL-10 production by TNF-alpha, IL-1 beta, IL-6, IL-8, and IFN-gamma in human colon carcinoma COLO205 cells. Northern analysis revealed a marked expression of IL-10 mRNA after stimulation by IL-6, and a marginal but significant expression by TNF-alpha, IL-1 beta or IFN-gamma. No IL-10 mRNA expression was observed when cells were untreated or incubated with IL-8. IL-10 in the culture supernatants showed good agreement with mRNA expression. In addition, IFN-gamma dose-dependently inhibited this IL-6-induced production of IL-10. MTT assay revealed that low dose IFN-gamma (1-10 ng/ml) had no effect on growth of COLO205 cells, but that high dose IFN-gamma (>100 ng/ml) significantly inhibited their proliferation. Northern analysis of COLO205 cells pretreated with IFN-gamma demonstrated that the IL-6R alpha chain was down-regulated. These results suggest that, in certain colon carcinoma cells, tumor-derived IL-10 production is directly regulated by systemic or local production of pro-inflammatory cytokines, such as IL-6 and IFN-gamma.