Cytokine sensitivity of metastatic human melanoma cell lines -simultaneous inhibition of proliferation and enhancement of gelatinase activity

The effect of a panel of cytokines on the proliferation and type IV collagenase production was studied in four melanoma cell lines of different origin, tumorigenicity and metastatic capacity. TGF-β, TNF-α and to a lesser extent, IL-lα exhibited antiproliferative effect on the cell lines, with some lines showing varying degree of resistance. The sensitivity did not correlate directly with the origin or the biological behavior of the tumor lines, suggesting that cytokine resistance of advanced stage melanoma cells may be relative. IL.-2- IL.-10 and IL.-12 displayed little or no effect on proliferation. The effect of cytokines on metalloproteinase production showed a cell line dependent pattern. Interestingly, those cytokines that exhibited the most pronounced antiproliferative activity, also proved most effective in stimulating collagenase secretion, often simultaneously, in the same line. The results indicate that pleiotropic cytokines can have positive and negative effects simultaneously on various steps of tumor progression.     

Malignant cell-specific gelatinase activity in human endometrial carcinoma.

BACKGROUND. The protease activity leading to degradation of the extracellular matrix was compared between human endometrial cancer and normal uterine endometrium. METHODS. Conditioned medium from tumor cells and normal endometrial cells was subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gel containing gelatin as a substrate. After electrophoresis, the gel was stained with Coomassie blue, and then the enzyme activity, expressed as the zone of dye clearing, was analyzed by densitometry. RESULTS. Densitometric analysis showed that all the endometrial cancers expressed a very high molecular weight enzyme activity (Mr 220,000), which was not detected in medium from normal endometrial cells. The analysis also showed that in endometrial cancer the activity of a Mr 92,000 enzyme was always superior to that of a Mr 64,000 enzyme, which was in contrast to the situation for normal endometrium. CONCLUSIONS. These results indicate that the expression of Mr 220,000 enzyme activity and the higher activity of the Mr 92,000 enzyme than the Mr 64,000 enzyme are involved in the malignant phenotype of native endometrial cancer.     

Expression of the oestrogen regulated pNR-2 mRNA in human breast cancer: relation to oestrogen receptor mRNA levels and response to tamoxifen therapy

The pNR-2 mRNA is regulated by oestrogens in cell lines established from metastatic human breast cancer cells. The levels of the pNR-2 and oestrogen receptor RNAs have been measured in 96 tumour samples from patients undergoing surgery for breast cancer. Oestrogen receptor mRNA was detected in 90% of the 60 primary breast tumour samples from patients not receiving endocrine therapy at the time of surgery, whereas the pNR-2 RNA was detected in 57%. In primary tumours the expression of pNR-2 was entirely dependent upon oestrogen receptor RNA expression. When the 60 primary tumours were considered, pNR-2 and oestrogen receptor mRNA levels were significantly correlated. There was no significant correlation for pNR-2 positive tumours. pNR-2 mRNA levels were similar in tumours of pre- and post-menopausal patients and were independent of tumour differentiation and nodal status. Oestrogen receptor and pNR-2 mRNA levels were also measured in 21 tumour samples from patients receiving primary tamoxifen therapy. Eleven of these had shown an objective response and a significantly larger number of tumours from these patients contained pNR-2 mRNA than from patients who did not respond (chi 2 = 6.08, P less than 0.025).     

mRNA levels for human nucleolar protein P120 in tumor and nontumor cells

A monoclonal antibody to a human tumor nucleolar 120 kD protein was developed by Freeman et al. (Cancer Res. 48: 1244-1251, 1988). Its complementary DNA (cDNA) has been isolated and sequenced (Fonagy et al., submitted). To determine the relative messenger RNA (mRNA) level for protein p120, cellular mRNA was extracted, slot-blotted onto nitrocellulose filters, and hybridized to radioactive p120 cDNA fragments. Human tumor cells contained 15-60 times more p120 mRNA than human term placenta. The rat Novikoff hepatoma ascites cell mRNA hybridized to the p120 cDNA probes, but the p120 monoclonal antibody did not react with the Novikoff hepatoma proteins. Novikoff hepatoma mRNA contained 8 times as much p120 mRNA as normal rat liver. As a control, a cDNA was used for protein B23, an abundant nucleolar protein; there were 3.5, 29, and 14 times more B23 mRNA than p120 mRNA in normal rat liver, Novikoff hepatoma ascites cells, and HeLa cells, respectively. Whereas the increased levels of the mRNA and protein B23 reflect increased activity of the nucleolus for any increment of nucleolar function, the increased levels of p120 mRNA and the p120 protein reflect the activity of the G1 phase of the cell cycle. The elevated level of p120 mRNA in tumors may reflect the heightened G1 cascade in transformed cells.     

人脑胶质瘤干细胞Fas/Fas-L mRNA表达的研究

目的:检测脑胶质瘤干细胞中Fas/Fas-L的mRNA表达水平,探讨其在脑胶质瘤凋亡以及免疫治疗中的意义。方法:从8例临床标本中培养出脑胶质瘤干细胞,并通过生物学特性和免疫细胞化学方法予以鉴定。然后提取脑胶质瘤干细胞以及相对应的原代脑胶质瘤细胞中的RNA,进行RealTime RT-PCR检测,测定Fas/Fas-L的mR-NA表达水平,并予统计学分析。结果:成功从8例临床标本中培养出脑胶质瘤干细胞并通过鉴定。定量检测mRNA表达显示,在脑胶质瘤干细胞中,Fas和Fas-L的mRNA表达均显著低于相应的原代培养的脑胶质瘤细胞,配对t检验显示,差异有统计学意义,P值均<0.001。结论:脑胶质瘤干细胞中Fas/Fas-L的mRNA表达水平低,可能会使以Fas为靶点的免疫治疗失败,从而导致肿瘤的复发。对Fas/Fas-L系统在脑胶质瘤治疗中的前景提出了疑问,但仍需进一步研究以明确判断。     

Antimetastatic activity of a novel mechanism-based gelatinase inhibitor

Matrix metalloproteinases (MMPs), and in particular gelatinases (MMP-2 and MMP-9), play a key role in cancer progression. However, clinical trials in which MMP inhibitors were tested in cancer patients have been disappointing. Whereas many reasons have been postulated to explain the failure of the clinical trials, lack of inhibitor selectivity was a major limitation. Thus, despite the consensus opinion that MMP-mediated proteolysis is essential for cancer progression and that certain MMPs represent important targets for intervention, effective and selective inhibition of those MMPs remains a major challenge in drug development. We previously reported the first mechanism-based MMP inhibitor, designated SB-3CT, which is a selective gelatinase inhibitor. Here we report that SB-3CT (5-50 mg/kg/d) is a potent inhibitor of liver metastasis and increases survival in an aggressive mouse model of T-cell lymphoma. This study shows that mechanism-based inhibition of gelatinases represents a novel approach to inhibitor design that promises to be a successful anticancer therapy.     

Functional blockade of Smad4 leads to a decrease in beta-catenin levels and signaling activity in human pancreatic carcinoma cells

In the last several years, many laboratories have tried to unravel the complex signaling mechanisms activated by TGF-beta(1) in transformed cells. Smad proteins are the principal mediators of the transforming growth factor beta (TGF-beta) response, but this factor can also activate Smad-independent pathways in different cell types. Our previous studies in murine keratinocytes led to the identification of a cooperation between oncogenic Ras and Smad4 inactivation during malignant progression. We further investigated the function of Smad4 in human pancreatic cancer, in which loss-of-function mutations affecting Smad4 occur with a 50% frequency. Expression of a dominant-negative Smad4 construct in the adenocarcinoma cell line PANC-1 led to increased ubiquitination and proteasomal degradation of beta-catenin. Moreover, loss of Smad4 abrogated beta-catenin-signaling activity and was associated with a reduction of the tumorigenic potential of PANC-1 cells in scid mice. Although the expression of the dominant-negative Smad4 blocked TGF-beta(1)/Smad2,3-signaling activity, the above-mentioned effects of Smad4 on beta-catenin stability were independent of the TGF-beta1/Smad2,3-signaling pathway. These findings provide evidence for a cross talk between Smad4 and the Wnt/beta-catenin pathway in pancreatic carcinoma cells, suggesting a new role for Smad4 as an attenuator of beta-catenin proteasomal degradation.     

Hyperthermia enhancement of radiation response and inhibition of recovery from radiation damage in human glioma cells

Three human glioma cell lines were tested for the effectiveness of hyperthermia and thermal radiosensitization. Thermal sensitization was evaluated from the perspective of increased radiosensitivity as well as inhibition of recovery from radiation damage. The three glioma cell lines tested showed large shoulders on the radiation survival curve and a large capacity for recovery of potentially lethal radiation damage. Hyperthermia caused radiosensitization in all three cell lines, which was primarily characterized by the reduction of the survival curve shoulder with moderate decreases in the survival curve slope. The radiosensitization was dependent on the time and temperature of the hyperthermia treatment. At 45 degree C for 60 min the shoulder of the radiation survival curve could be completely eliminated and the degree of enhanced cell killing at the 2 Gy level ranged from factors of 10 to 20 under the various conditions. When hyperthermia was given to cells which were irradiated and then plated immediately, or delayed for 8 h before plating to allow recovery, hyperthermia was found to cause radiosensitization under both conditions. In addition, when the hyperthermia dose was increased the difference between the immediate plating and the delayed plating survival curve decreased and for 45 degrees C for 60 min this difference was completely eliminated, concomitantly with the elimination of the survival curve shoulder. These data indicate that hyperthermia may play a role in radiosensitization for the treatment of human glioma.     

Hyperthermia enhancement of radiation response and inhibition of recovery from radiation damage in human glioma cells

Three human glioma cell lines were tested for the effectiveness of hyperthermia and thermal radiosensitization. Thermal sensitization was evaluated from the perspective of increased radiosensitivity as well as inhibition of recovery from radiation damage. The three glioma cell lines tested showed large shoulders on the radiation survival curve and a large capacity for recovery of potentially lethal radiation damage. Hyperthermia caused radiosensitization in all three cell lines, which was primarily characterized by the reduction of the survival curve shoulder with moderate decreases in the survival curve slope. The radiosensitization was dependent on the time and temperature of the hyperthermia treatment. At 45°C for 60 min the shoulder of the radiation survival curve could be completely eliminated and the degree of enhanced cell killing at the 2 Gy level ranged from factors of 10 to 20 under the various conditions. When hyperthermia was given to cells which were irradiated and then plated immediately, or delayed for 8 h before plating to allow recovery, hyperthermia was found to cause radiosensitization under both conditions. In addition, when the hyperthermia dose was increased the difference between the immediate plating and the delayed plating survival curve decreased and for 45°C for 60 min this difference was completely eliminated, concomitantly with the elimination of the survival curve shoulder. These data indicate that hyperthermia may play a role in radiosensitization for the treatment of human glioma.     

Interferon and natural killer activity in multiple myeloma. Lack of correlation between interferon-induced enhancement of natural killer activity and clinical response to human interferon-alpha

Natural killer (NK) activity of peripheral lymphocytes was measured in 39 patients with multiple myeloma prior to and during interferon (IFN) therapy. NK activity increased in a majority of patients following the first injection of IFN and remained at an increased level during 1 year of therapy. Lower doses of IFN seemed to induce a greater increase in NK activity than higher doses. No correlations could be observed between the response of the tumors to IFN therapy and pretreatment levels of NK activity, IFN-induced enhancement of NK activity in vitro or IFN-induced enhancement of NK activity in vivo.     

Reductions in gamma-globin mRNA levels restricted to the cytoplasm of 5-fluorouridine treated K-562 human erythroleukemia cells

RNA synthesis in K-562 human erythroleukemia cells was markedly curtailed by exposure to the uridine analogue 5-fluorouridine (FUrd). The inhibition of ribosomal RNA synthesis was accompanied by rapid declines in the steady-state levels of several, but not all, mRNAs, including gamma-globin mRNA. In this report, we demonstrate that gamma-globin mRNA species were decreased by as much as 40% within 2 hr of exposure to micromolar concentrations of FUrd. The decline in gamma-globin mRNA occurred at a rate that outstripped the normal rate of degradation of this mRNA by a factor of 25. The decline in cytoplasmic mRNA was not mirrored in the nucleus; northern blotting revealed that pre-mRNA levels were not reduced. Nuclease protection analyses of precursors from FUrd treated and untreated control cells did not reveal any qualitative differences. Thus, the decrease was not accounted for by drug-induced inhibition of new gamma-globin mRNA synthesis or misincorporation but must have been due to an FUrd-induced increase in gamma-globin mRNA degradation. Drug-induced instability of RNA was not a generalized feature of FUrd exposure, since neither beta-actin mRNA nor cytoplasmic rRNA, whose stabilities in untreated cells are similar to that of gamma-globin mRNA, were affected. Furthermore, the instability of gamma-globin mRNA did not decrease globin protein levels, presumably because the stability of the protein was not altered. The mechanism by which specific increased degradation of gamma-globin mRNA occurred is unknown, but it may have been due to the activation of cytoplasmic endonuclease.     

Tumor degeneration by human embryonic fibroblasts and its enhancement by interferon

KB cells from a human nasopharyngeal tumor were cocultivated with human embryonic fibroblasts (HF 8101 cells); 7 to 14 days after incubation, "spongy degeneration"-like changes developed in the target cell-growing area. These changes developed in other target cells [HeLa cells from human cervical cancer, human hepatoma cells (PLC/PRF/5), and human amnion FL cells] cocultured with several kinds of human embryonic fibroblasts [HF 8101 cells, HF 8103 cells, and HEL cells]; however, HF 8101 cells did not cause degenerative changes in murine L929 cells. The degenerative changes were enhanced by treatment with human leukocyte interferon or human fibroblast interferon at a dose of 1,000 or 10,000 IU/ml, but there was no significant difference in the enhancing effect between human leukocyte and human fibroblast interferons. Mouse L929 interferon did not enhance the degenerative changes in KB cells caused by HF 8101 cells. It was concluded that human fibroblasts caused the degenerative changes in the human tumor cells and the continuous cell line and that the changes were enhanced by treatment with either human leukocyte interferon or human fibroblast interferon.     

Collagenase stimulation in cocultures of human fibroblasts and human tumor cells

Cocultures of human fibroblasts with 3 different human tumor cell lines exhibited high levels of collagenase activity against type I collagen compared to individual cultures of each cell type. For LX-1 lung carcinoma cells maximum stimulation occurred at a ratio of tumor cells to fibroblasts of 0.5:1. These cocultures contained 15-50 times more collagenase activity than the sum of the individual cultures. Conditioned medium from the LX-1 cells stimulated the fibroblasts to an extent similar to that obtained in the cocultures. Conditioned medium from the fibroblasts did not stimulate the tumor cells. Thus human tumor cells secrete a factor(s) which stimulates collagenase production by human fibroblasts.     

Tumor-induced alterations in tissue lipoprotein lipase activity and mRNA levels

To elucidate the mechanisms of hypertriglyceridemia observed in the tumor-bearing rat, tissue lipoprotein lipase (LPL) activity and LPL mRNA levels were examined in the fed and fasted states at different degrees of tumor burden and after tumor removal. LPL activity in the epididymal fat pad and cardiac muscle in the 24-h-fasted rats was significantly decreased with increasing tumor burden (r = -0.53, P less than 0.05 and r = -0.72, P less than 0.01, respectively). Tumor removal completely reversed these changes. In contrast, no change in LPL activity was detected in the fed state since food intake stimulated LPL activity to the same extent in both tumor-bearing (TBR) and control rats. LPL activity in the diaphragm and skeletal muscle was only marginally altered in TBR, as compared to controls. LPL mRNA from the epididymal fat pad and cardiac muscle migrated to the same site on agarose gel and hybridized to a LPL-specific complementary DNA probe. The decline in LPL activity in epididymal fat pad observed in TBR was associated with a decrease in LPL mRNA levels. In contrast, there was no significant difference in LPL mRNA levels in cardiac muscle between the two groups despite significantly suppressed enzyme activity in tumor bearers. This study provides evidence that hypertriglyceridemia in TBR is due in part to tumor-dependent suppression of adipose and cardiac LPL activity in the fasted state, which is stimulated by the presence of tumor. Unlike cardiac LPL, the tumor-induced changes in adipose LPL activity are regulated at the mRNA level in this tumor model.     

Gene amplification of human cytidine deaminase proviral cDNA and increased levels of its mRNA produces enhanced drug resistance to cytosine arabinoside in retroviral-transduced murine fibroblasts

Hematopoietic toxicity is one of the major problems that limits the effectiveness of many antineoplastic drugs. One approach to overcome this problem is to confer chemoresistance to the hematopoietic cells by gene transfer of drug resistance genes. Human cytidine deaminase (CD) inactivates the cytosine nucleoside analogues, such as cytosine arabinoside (ARA-C), by deamination. We have reported previously that retroviral-mediated gene transfer of CD conferred drug resistance to ARA-C in murine cells. One of the major problems in the use of these vectors is to obtain adequate and prolonged expression of the transferred gene to produce a therapeutic effect in the transduced cells. The objective of this investigation was to determine if it is possible to increase the expression of CD proviral DNA in transduced murine fibroblast cells. We observed that by the use of continuous exposure to increasing concentrations of ARA-C it was possible to enhance drug resistance in the transduced cells. This drug resistance was found to be associated with increases in CD enzyme activity and CD proviral mRNA and by amplification of the proviral CD gene.     

Differential growth response of normal human diploid fibroblasts and in vitro transformed human fibroblasts in serum-free defined culture medium

Two neoplastic human cell lines, WI-38 CT-1 and SUSM-1, which were transformed in vitro with gamma rays and 4-nitroquinoline 1-oxide, respectively, grew continuously in a serum-free defined medium. The defined medium used was a 1:1 mixture by volume of Dulbecco's modified Eagle's medium and Ham's F12 (DF) supplemented with 0.1% bovine serum albumin fraction V, 10 micrograms/ml of transferrin, 1 microgram/ml of insulin, and 5 micrograms/ml of oleic acid. In the case of SUSM-1, 100 micrograms/ml of fetuin were added to cultures when the cells were subcultured. Under these conditions the growth rates of the two transformed human cell lines were almost the same as those in a DF medium containing 10% fetal bovine serum (FBS). In addition, the defined medium permitted the cells to grow indefinitely without a lag period when they were transferred from serum-containing medium into this defined medium, indicating that no selection or adaptation of the cells had occurred. Interestingly, these cell lines did not require for their growth any polypeptide growth factors such as epidermal, platelet-derived or fibroblast growth factors. On the other hand, the control WI-38 cells stopped growing in the defined medium after about 2 divisions. Another control normal cell strain of fibroblasts derived from a human embryo showed a decreased growth rate in the defined medium as compared with that in the DF medium with 10% FBS. These results suggest that the defined medium described here is useful for the selective growth of human cells transformed in vitro after treatment with carcinogens from an untransformed cell population. In addition, the defined medium for transformed human cells should contribute to studies on their growth mechanisms.     

Selenomethionine induction of DNA repair response in human fibroblasts

Selenium compounds have a long history in chemoprevention of mammary and colon cancers in rodent models. Selenium compounds are in current clinical trials, having shown promise in prevention of prostate and other human cancers. In human tissues, it has been estimated that each cell sustains approximately 10 000 potentially mutagenic (if not repaired) lesions per day due to endogenous DNA damage. Almost no studies have addressed the potential for selenium compounds to induce DNA repair, a potential mechanism for their cancer-preventive actions. We show that selenium in the form of selenomethionine induces a DNA repair response in normal human fibroblasts in vitro, and protects cells from DNA damage. We show a possible mechanism for the inducible DNA repair response, in which enhanced repair complex formation was observed in selenomethionine-treated cells.     

Selective modulation of glutathione levels in human normal versus tumor cells and subsequent differential response to chemotherapy drugs

Cellular glutathione (GSH) levels were found to be 7-fold higher in a human lung adenocarcinoma cell line (A549) than in a normal human lung fibroblast line (CCL-210). Differential modulation of cellular GSH was explored in these cell lines by (a) stimulation of GSH synthesis by oxothiazolidine-4-carboxylate (OTZ) and (b) inhibition of GSH synthesis by buthionine sulfoximine (BSO). In the tumor cell line, OTZ treatment had no effect; however, GSH levels of 140-170% of control were achieved in the normal fibroblast line. With BSO, the normal cell line was depleted of GSH at a faster relative rate than with the tumor line. Within 7 h, 5% GSH remained in the CCL-210 line while approximately 40% GSH remained in the A549 line. Survival response of normal versus tumor cell lines to selected chemotherapy drugs was compared following modulation of GSH levels. OTZ pretreatment of the A549 line provided no protection to a 1-h exposure to melphalan, cisplatin, or bleomycin; however, OTZ pretreatment of CCL-210 elevated GSH and provided protection to melphalan, cisplatin, and bleomycin (protection ratios at 5% survival of 1.2, 1.4, and 1.4, respectively). Neocarzinostatin toxicity in the normal CCL-210 line pretreated with BSO was greatly reduced (protection ratio at 50% survival = 5.0). The same BSO treatment to A549 cells (40% GSH remaining) yielded a similar survival curve to control cells. These studies demonstrate that selective differential chemotherapy responses of normal versus tumor cells is possible by manipulating the GSH synthetic cycle. Should basic phenotypic differences with regard to reductive capacity exist in vivo, such manipulation in GSH levels might yield a therapeutic gain for carefully selected chemotherapy drugs.