Estrogen inhibits paclitaxel-induced apoptosis via the phosphorylation of apoptosis signal-regulating kinase 1 in human ovarian cancer cell lines

The influence of postoperative estrogen replacement therapy on the sensitivity of ovarian cancer to paclitaxel remains elusive. We examined whether estrogen affects paclitaxel-induced apoptosis in the Caov-3 human ovarian cancer cell line, which expresses estrogen receptor. 17beta-Estradiol (E2) significantly reversed the paclitaxel-induced apoptosis and reduction of cell viability, and a highly selective estrogen receptor antagonist, ICI182,780, and a phosphatidylinositol 3-kinase inhibitor, LY294002, attenuated the reversal effect of E2 on paclitaxel-induced apoptosis and reduction of cell viability. E2 significantly induced the phosphorylation of Akt. Akt and apoptosis signal-regulating kinase 1 (ASK1) were physically associated, and E2 induced the phosphorylation of ASK1 at serine-83, which is a consensus Akt phosphorylation site. We confirmed a previous report showing that paclitaxel induces cell damage via the ASK1-c-Jun N-terminal protein kinase (JNK) cascade. E2 inhibited the paclitaxel-induced JNK activation, and the E2-induced inhibition of the paclitaxel-induced JNK activation was attenuated in cells treated with either ICI182,780 or LY294002 or transfected with ASK1S83A, in which a consensus Akt phosphorylation site at serine-83 was converted to alanine. The inhibitory effect of E2 on the paclitaxel-induced reduction of cell viability and apoptosis was diminished in cells transfected with ASK1S83A. These results indicate that E2 inhibits paclitaxel-induced cell damage by inhibiting JNK activity via phosphorylation of Akt-ASK1. Thus, treatment of ovarian cancer with paclitaxel might be less effective in the setting of postoperative estrogen replacement therapy.     

BRAFV600E mutation is associated with preferential sensitivity to mitogen-activated protein kinase kinase inhibition in thyroid cancer cell lines

CONTEXT: Mutually exclusive mutations of RET, RAS, or BRAF are present in about 70% of papillary thyroid carcinomas, whereas only the latter two are seen in poorly differentiated and anaplastic cancers. Although the signal output common to these oncoproteins is ERK, a recent report showed that only BRAF mutations consistently predicted responsiveness to MAPK kinase (MEK) inhibitors. OBJECTIVES: Here we investigated whether sensitivity to MEK inhibition was determined by oncogene status in 13 human thyroid cancer cell lines: four with BRAF mutations, four RAS, one RET/PTC1, and four wild type. RESULTS: Growth of BRAF (+) cells was inhibited by the MEK antagonist PD0325901 with an IC(50) of less than 5 nm. By contrast, RAS, RET/PTC1, or wild-type cells had IC(50) of 4 nm to greater than 1000 nm. Sensitivity was not predicted by coexisting mutations in PIK3CA or by PTEN status. Similar effects were obtained with the MEK inhibitor AZD6244. PD0325901 induced a sustained G1/S arrest in BRAF (+) but not BRAF (-) lines. PD0325901 was equipotent at inhibiting pERK1/2 after 2 h, regardless of genetic background, but pERK rebounded at 24 h in most lines. MEK inhibitor resistance was associated with partial refractoriness of pERK to further inhibition by the compounds. AZD6244 was more potent at inhibiting growth of NPA (BRAF +) than Cal62 (KRAS +) xenografts. CONCLUSION: Thyroid cancers with BRAF mutation are preferentially sensitive to MEK inhibitors, whereas tumors with other MEK-ERK effector pathway gene mutations have variable responses, either because they are only partially dependent on ERK and/or because feedback responses elicit partial refractoriness to MEK inhibition.     

Growth factors change nuclear distribution of estrogen receptor-alpha via mitogen-activated protein kinase or phosphatidylinositol 3-kinase cascade in a human breast cancer cell line

In the present study, to examine the dynamic changes in the localization of nuclear estrogen receptor (ER)alpha induced by growth factors, we used time-lapse confocal microscopy to directly visualized ERalpha fused with green fluorescent protein (GFP-ERalpha) in single living cells treated with epidermal growth factor (EGF) or IGF-I. We observed that 17beta-estradiol (E2) changed the normally diffuse distribution of GFP-ERalpha throughout the nucleoplasm to a hyperspeckled distribution within 10 min. Both EGF and IGF-I also changed the nuclear distribution of GFP-ERalpha, similarly to E2 treatment. However, the time courses of the nuclear redistribution of GFP-ERalpha induced by EGF or IGF-I were different from that induced by E2 treatment. In the EGF-treated cells, the GFP-ERalpha nuclear redistribution was observed at 30 min and reached a maximum at 60 min, whereas in the IGF-I-treated cells, the GFP-ERalpha nuclear redistribution was observed at 60 min and reached a maximum at 90 min. The EGF-induced redistribution of GFP-ERalpha was blocked by pretreatment with a MAPK cascade inhibitor, PD98059, whereas the IGF-I-induced redistribution of GFP-ERalpha was blocked by pretreatment with a phosphatidylinositol 3-kinase inhibitor, LY294002. Analysis using an activation function-2 domain deletion mutant of GFP-ERalpha showed that the change in the distribution of GFP-ERalpha was not induced by E2, EGF, or IGF-I treatment. These data suggest that MAPK and phosphatidylinositol 3-kinase cascades are involved in the nuclear redistribution of ERalpha by EGF and IGF-I, respectively, and that the activation function-2 domain of ERalpha may be needed for the nuclear redistribution of ERalpha.     

Estradiol hypersensitivity and mitogen-activated protein kinase expression in long-term estrogen deprived human breast cancer cells in vivo

Women with breast cancer who have responded to initial hormonal therapy frequently experience additional remissions upon further endocrine manipulation. We postulate that hypersensitivity to estradiol (E2) may serve as a mechanistic explanation for these secondary responses. We previously provided evidence of hypersensitivity using an in vitro breast cancer model system and demonstrated the role of mitogen-activated protein kinase (MAP kinase) in the process of adaptation to long-term estradiol deprivation. In the present study, we wished to demonstrate that hypersensitivity to E2 could occur under more complex in vivo conditions and that MAP kinase activation is enhanced under these circumstances. We used an MCF-7 breast cancer model system involving long-term estradiol deprived (LTED) cells to produce xenografts in nude mice and an E2 clamp method to precisely control sex steroid levels. The E2 clamp was designed to maintain plasma E2 at a series of doubling doses from 1.25 pg/ml to 20.0 pg/ml in oophorectomized nude mice. As evidence of the validity of the clamp method, a uterine weight bioassay revealed an excellent, linear dose-response relationship between the predicted level of plasma E2 and stimulation of uterine weight. As evidence of hypersensitivity, we found that LTED xenograft tumors grew to a greater extent than wild-type in response to E2 concentrations of 1.25 pg/ml (P = 0.003) and 2.5 pg/ml (P = 0.0002). At the 10.0 and 20.0 pg/ml plasma concentrations, the LTED tumors were stimulated to a lesser extent than the wild-type. This pattern of increased growth at lower concentrations and reduced growth vs. the wild-type at higher concentrations mimics closely the pattern seen for LTED cells in vitro. All LTED cell tumors exhibited enhanced activation of MAP kinase ranging from 18 to 25%, and E2 did not increase this further. In contrast, E2 caused a linear increase in the percentage of activated MAP kinase positive cells (P < 0.0001) in wild-type tumors from basal levels of 2.66% to maximal levels of 6.40%. These observations suggest a dynamic interplay whereby activation of MAP kinase renders cells more sensitive to the proliferative effects of E2. The precise mechanisms for this interplay are unknown but, when further understood, could potentially provide insight into approaches to prevent the evolution of tumors to a hormone insensitive state.     

Differential sensitivity of human endometrial carcinoma cells with different PTEN expression to mitogen-activated protein kinase signaling inhibits and implications for therapy

Purpose  

Prior studies in different tumor models have shown that, under conditions of PTEN deficiency, the mitogen-activated protein kinase (MAPK) signaling pathway appear to play a major role in the tumor cell’s proliferative and survival pathway, and that pharmacological inhibition of this pathway results in tumor growth inhibition. This study aimed to explore whether sensitivity to p38MAPK inhibitors are specifically due to status of PTEN in endometrial cancer cells.     

Modulation of cisplatin sensitivity by taxol in cisplatin-sensitive and -resistant human ovarian carcinoma cell lines

Purpose: The aim of this study was to determine whether taxol can circumvent cisplatin resistance, using a KF28 cell line derived from human ovarian carcinoma and a cisplatin-resistant line, KFr13, derived from the parental cell line, KF28, and taxol-resistant cell lines, KF28_TX and KFr13_TX, derived from the respective parental counterpart. Methods: KF28 is a single-cell clone of the human ovarian carcinoma cell line KF. The cisplatin-resistant KFr13 subline was established by repeated exposure of the parent KF28 cell line to escalating doses of cisplatin. Similarly, KF28_TX and KFr13_TX were established by repeated exposure of the KF28 and KFr13 cell lines to escalating doses of taxol. A cytotoxicity assay was performed using a crystal violet staining method. Platinum and taxol accumulation were assayed by atomic absorption and reverse-phase high-performance liquid chromatography. The quantitative assay of MDR1 mRNA used polymerase chain reaction. Results: KFr13 cells were about 4.8-fold more resistant to cisplatin and about 1.8-fold more sensitive to taxol than were KF28 cells. When taxol resistance was induced in KF28 and KFr13 cells, sensitivity to cisplatin rose about 1.3- and 1.6-fold respectively. Elevation of sensitivity was correlated with platinum uptake by both KF28_TX and KFr13_TX cells. Expression of multidrug resistance (MDR1) mRNA, which was not observed in KF28 and KFr13 cells, was observed after induction of taxol resistance. Conclusions: These results may suggest rational therapeutic strategies for patients with cisplatin-resistant or refractory ovarian carcinoma. Received: 1 April 1999 / Accepted: 13 May 1999     

Dehydroepiandrosterone improves murine osteoblast growth and bone tissue morphometry via mitogen-activated protein kinase signaling pathway independent of either androgen receptor or estrogen receptor

Dehydroepiandrosterone (DHEA) may be a promising agent for postmenopausal osteoporosis (PMO), but its mechanism to modulate osteoblasts (OBs) is yet to be explained. To elucidate the effects of DHEA treatment on the ovariectomized (OVX) mice and its mechanisms, we evaluated the morphology of mice bone tissue and expression of proliferating cell nuclear antigen (PCNA) in the vertebrae-derived OB after having treated the OVX animals with DHEA. The results showed that DHEA administration increased the expression of PCNA in OB and changed the bone tissue morphometry of the PMO model. To further investigate this mechanism, the OB was isolated from neonatal mice calvariae by the enzyme-digested assay, exposed to DHEA, and then analyzed for ultrastructure, DNA content, early apoptotic cells, and phosphorylation of extracellular signal-regulated kinase 1/2. It was found that DHEA promoted proliferation and inhibited apoptosis of OB significantly, via mitogen-activated protein kinase signaling pathway independent of either androgen receptor or estrogen receptor, suggesting that it may exert roles via a DHEA-specific receptor directly, not by way of conversion to androgens or estrogens.     

Regulation of ovarian cancer cell viability and sensitivity to cisplatin by progesterone receptor membrane component-1

CONTEXT: Progesterone (P4) influences ovarian cancer cells by an unknown mechanism. OBJECTIVE: The objective was to determine whether P4 acts through progesterone receptor membrane component-1 (PGRMC1) in ovarian cancers. DESIGN, SETTING AND PATIENTS: Archival tissue and cDNA provided by OriGene were used for expression studies. In vitro experiments were conducted with Ovcar-3 cells. MAIN OUTCOME MEASURES: PCR, Western blot, and immunohistochemistry were used to measure expression of PGRMC1 and nuclear progesterone receptor (PGR). PGRMC1's role in regulating the viability of ovarian cancers was assessed by overexpressing PGRMC1, depleting PGRMC1 using small interfering RNA, and attenuating PGRMC1's action with a blocking antibody. Apoptosis was determined by 4',6'-diamino-2-phenylindole staining. RESULTS: PGRMC1 mRNA increased and PGR mRNA decreased in advanced stages of ovarian cancer. Unlike PGR, PGRMC1 was expressed in virtually every cancer cell within the tumor. A similar relationship between PGRMC1 and PGR was observed in Ovcar-3 cells. In these cells P4 suppressed apoptosis induced by either serum withdrawal or cisplatin (CDDP). Moreover, in the presence of P4, the following occurs: 1) overexpression of PGRMC1 reduces the effectiveness of CDDP, 2) depletion of PGRMC1 with small interfering RNA enhances the effects of CDDP, and 3) PGRMC1 antibody treatment increases the apoptotic response to CDDP. CONCLUSIONS: These findings indicate that PGRMC1 plays an important role in promoting ovarian cancer cell viability and that attenuating PGRMC1's action makes the ovarian cancer cells more sensitive to CDDP. These data suggest that targeted depletion of PGRMC1 could be useful as an adjunct to CDDP therapy.     

Functional expression of bombesin receptor in most adult and pediatric human glioblastoma cell lines; role in mitogenesis and in stimulating the mitogen-activated protein kinase pathway

Functional bombesin receptors were identified in most human glioblastoma cell lines examined (85% of lines). Bombesin stimulated the release of intracellular Ca²⁺ in human adult (U-373MG, D-247MG, U-118MG, U-251MG, D-245MG, U-105MG, D-54MG, A-172MG, and D-270MG lines) and pediatric (SJ-S6 and SJ-G2 lines) glioblastoma cell lines. Stimulation of the glioblastoma cell line U-373MG with bombesin or gastrin-releasing peptide (GRP) induced mitogenesis, measured by [³H]thymidine incorporation into DNA, and stimulated the tyrosine phosphorylation of the mitogen-activated protein (MAP) kinases (Erk1 and Erk2). The stimulation of the MAP kinase phosphorylation in U-373MG cells was time- and peptide concentration-dependent. Both bombesin and GRP showed similar potencies in stimulation of intracellular Ca²⁺ release and activation of the MAP kinase pathway in U-373MG cells, whereas neuromedin B (NMB) peptide was less potent. Bombesin and GRP induced the release of cytosolic Ca²⁺ in a concentration-dependent manner. Because bombesin and GRP were more potent than NMB peptide in increasing the cytosolic Ca²⁺ levels in U-373MG cells, we concluded that the BB₂ subtype (also known as GRP-preferring receptor subtype) of the bombesin receptor is expressed in this cell line. The bombesin receptor antagonist ([Leu¹³-ψ(CH₂NH)Leu¹⁴]bombesin) blocked bombesin induced Ca²⁺ release and attenuated MAP kinase activation in U-373MG cells demonstrating that bombesin is acting through a receptor-dependent mechanism. This study indicates that functional bombesin receptors are widely expressed in human glioblastoma cell lines.     

Insulin inhibits angiotensinogen gene expression via the mitogen-activated protein kinase pathway in rat kidney proximal tubular cells.

The present study aimed to investigate the molecular mechanism(s) of insulin action on angiotensinogen (ANG) secretion and gene expression in kidney proximal tubular cells exposed to high levels of glucose. Immortalized rat proximal tubular cells (IRPTC) were cultured in monolayer. The levels of rat ANG and ANG messenger RNA in the IRPTC were quantified by a specific RIA for rat ANG (RIA-rANG) and by an RT-PCR assay. Insulin inhibited the stimulatory effect of a high level of glucose (25 mM) and phorbol 12-myristate 13-acetate, an activator of protein kinase C) on the secretion of ANG and the expression of the ANG messenger RNA in IRPTC. This inhibitory action of insulin on the ANG secretion and gene expression was blocked by PD98059 (an inhibitor of mitogen-activated protein kinase kinase) but not by Wortmannin (an inhibitor of phosphatidylinositol-3-kinase). PD98059 was effective in inhibiting the phosphorylation of MEK 1/2 and p44/42 MAP kinase in IRPTC stimulated by insulin. These studies demonstrate that insulin prevents the stimulatory effect of high levels of glucose on the expression of the renal ANG gene in IRPTC, at least in part, via the MAPK kinase signal transduction pathway, subsequently inhibiting the activation of the local renal renin-angiotensin system.     

胰腺癌细胞中血管紧张素Ⅱ1型受体mRNA和蛋白的表达

目的　近年来的研究显示 ,血管紧张素Ⅱ 1型受体 (AT1)具有促肿瘤细胞生长和促进肿瘤血管生成作用 ,运用其拮抗剂则具有抑制肿瘤生长作用。对胰腺癌细胞中AT1mRNA和蛋白的表达进行探讨。方法　应用SW 1990、PaTu8988s和PANC 1胰腺癌细胞系。RT PCR方法检测胰腺癌细胞系中AT1mRNA的表达 ;分别用免疫细胞化学染色与SDS 聚丙烯酰胺凝胶电泳 (SDS PAGE)检测胰腺癌细胞系中蛋白的表达。结果　在 3个胰腺癌细胞系中均有AT1mRNA的表达 ;AT1蛋白表达位于细胞膜和细胞质内 ;蛋白电泳也证实 3个人胰腺癌细胞系中均有相对分子质量为 4 4× 10 3 的AT1蛋白的表达。结论　AT1在胰腺癌细胞生长中似乎发挥重要作用 ,抑制AT1可能是一种有用的治疗策略。     

Inhibition of protein-kinase-C — dependent cell proliferation of human lung cancer cell lines by the dihydropyridine dexniguldipine

The dihydropyridine, dexniguldipine hydrochloride (B859-35), has shown therapeutic activity in experimentally induced neuroendocrine hamster lung tumors and demonstrated antiproliferative effects in a mammary cancer cell line via inhibition of Ca²⁺ calmodulin. Studies in NIH 3T3 fibroblasts have provided evidence that dexniguldipine may also inhibit protein kinase C (PCK). In this study, we have tested the hypothesis that dexniguldipine may inhibit the proliferation of lung cancer cells in response to autocrine or exogenous activation of PKC. Using a panel of human lung cancer cell lines, we show that dexniguldipine is a potent inhibitor of mitogenic signal transduction pathways dependent on PKC activation in several small-cell and non-small-cell lung cancer cell lines while it failed to inhibit cyclic-AMP-dependent cell proliferation.Supported in part by grant R55 CA 51211-01A1 with the National Cancer Institute     

Celecoxib inhibits proliferation and induces apoptosis via prostaglandin E2 pathway in human cholangiocarcinoma cell lines

AIM: To evaluate the roles and mechanisms of celecoxib in inducing proliferation inhibition and apoptosis of human cholangiocarcinoma cell lines. METHODS: Cyclooxygenase-2-overexpressing human cholangiocarcinoma cell line QBC939 and cyclooxygenase2-deficient human cholangiocarcinoma cell line SK-CHA-1were used in the present study. The anti-proliferative effect was measured by methabenzthiazuron (MTT) assay;apoptosis was determined by transferase-mediated dUTP nick end labeling (TUNEL) detection and transmission electron microscopy (TEN). Cell cycle was analyzed by flow cytometry (FCM). The PGE2 levels in the supernatant of cultured cholangiocarcinoma cells were quantitated by enzyme-linked immunoabsordent assay (ELISA). RESULTS: Celecoxib suppressed the production of PGE2and inhibited the growth of QBC939 cells. Celecoxib at 10,20, and 40 μmol/L inhibited PGE2 production by 26 %,58 %, and 74 % in QBC939 cells. The PGE2 level was much lower constitutively in SK-CHA-1 cells (18.6±3.2)compared with that in QBC939 (121.9±5.6) cells (P＜0.01)and celecoxib had no significant influence on PGE2 level in the SK-CHA-1 cells. The PGE2 concentration in SK-CHA-1cells also reduced but not significantly after treatment with celecoxib. The PGE2 concentration in SK-CHA-1 cells was (16.5±2.9) ng/well, (14.8±3.4) ng/well, (13.2±2.0) ng/well and (12.6±3.1) ng/well respectively, when pre-treated with 1 Jmol/L, 10 Jmol/L, 20 Jmol/L and 40 Jmol/L of celecoxib for 48 h (P＞0.05, vs control). The anti-proliferation effect of celecoxib (20 Jmol/L) on QBC939 cells was time-dependent,it was noticeable on day 2 (OD490=0.23±0.04) and became obvious on day 3 (OD490=0.31±0.07) to day 4 (OD490=0.25±0.06), and the OD490 in the control group (day 1)was 0.12±0.03 (P＜0.01, vscontrol). The anti-proliferation effect of celecoxib could be abolished by the addition of 200 pg/mL PGE2. The proliferation of SK-CHA-1 cells was inhibited slightly by celecoxib, the cell density OD490 in the presence of celecoxib and in control group was 0.31±0.04 and 0.42±0.03 respectively on day 2 (P＞0.05), 0.58±0.07 and 0.67±0.09 respectively on day 3 (P＞0.05), and 0.71±0.08 and 0.78±0.06 respectively on day 4 (P＞0.05). Celecoxib induced proliferation inhibition and apoptosis by G1-S cell cycle arrest: the percentage of QBC939 cells in G0-G1 phase after treatment with 40 Jmol/L (74.66±6.21) and 20 Jmol/L (68.63±4.36) celecoxib increased significantly compared with control cells (54.41±5.12, P＜0.01). The percentage of SK-CHA-1 cells in G0-G1 phase after treatment with various concentrations of celecoxib didn't change significantly compared with control cells. The TUNEL index was much higher in QBC939 cells treated with 20 Jmol/L celecoxib for 2 d (0.063±0.018) and for 4 d (0.102±0.037) compared with control cells (0.017±0.004, P＜0.01). CONCLUSION: The currentin vitro study indicates that inhibition of proliferation and induction of apoptosis in human cholangiocarcinoma cells by cyclooxygenase-2 specific inhibitor celecoxib may involve in COX-dependent mechanisms and PGE2 pathway. Celecoxib as a chemopreventive and chemotherapeutic agent might be effective primarily on COX2-expressing cholangiocarcinoma.     

Increased expression of mitogen-activated protein kinase and its upstream regulating signal in human gastric cancer

AIM: To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological parameters.METHODS: Western blot was used to measure the expression of extracellular signal-regulated kinase (ERK)-1, ERK-2, ERK-3, p38 and mitogen or ERK activated protein kinaseMEK-1 proteins in surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 42 patients. Immunohistochemistry was employed for their localization.RESULTS: Compared with normal tissues, the protein levels of ERK-1 (integral optical density value 159526&#177;65760 vs 122807&#177;65515, P=0.001), ERK-2 (168471&#177;95051 vs 120469&#177;72874, P&lt;0.001), ERK-3 (118651&#177;71513 vs 70934&#177;68058, P&lt;0.001), P38 (104776&#177;51650 vs 82930&#177;40392, P=0.048) and MEK-1 (116486&#177;45725 vs 101434&#177;49387, P=0.027) were increased in gastric cancer tissues. Overexpression of ERK-3 was correlated to TNM staging [average ratio of integral optic density (IOD)tumor:IODnormal in TNM Ⅰ, Ⅱ, Ⅲ, Ⅳ tumors was 1.43&#177;0.34, 5.08&#177;3.74, 4.99&#177;1.08, 1.44&#177;1.02, n=42, P=0.023] and serosa invasion (4.31&#177;4.34 vs 2.00&#177;2.03, P=0.037). In poorly differentiated cancers (n=33), the protein levels of ERK-1 and ERK-2 in stage Ⅲ and Ⅳ tumors were higher than those in stage Ⅰ and Ⅱ tumors (2.64&#177;3.01 vs 1.01&#177;0.33, P=0.022; 2.05&#177;1.54 vs 1.24&#177;0.40, P=0.030). Gastric cancer tissues with either lymph node involvement (2.49&#177;2.91 vs 1.03&#177;0.36, P=0.023; 1.98&#177;1.49 vs 1.24&#177;0.44, P=0.036) or serosa invasion (2.39&#177;2.82 vs 1.01&#177;0.35, P=0.022; 1.95&#177;1.44 vs 1.14&#177;0.36, P=0.015) expressed higher protein levels of ERK-1 and ERK-2. In Borrmann Ⅱ tumors, expression of ERK-2 and ERK-3 was increased comparedwith Borrmann Ⅲ tumors (2.57&#177;1.86 vs 1.23&#177;0.60, P=0.022; 5.50&#177;5.05 vs 1.83&#177;1.21, P=0.014). Borrmann Ⅳ tumors expressed higher p38 protein levels. No statistically significant difference in expression of MAPKs was found when stratified to tumor size or histological grade (P&gt;0.05). Protein levels of ERK-2, ERK-3 and MEK-1 in metastatic lymph nodes were 2-7 folds higher than those in adjacent normal mucosa. The immunohistochemistry demonstrated that ERK-1, ERK-2, ERK-3, p38 and MEK-1 proteins were mainly localized in cytoplasm. The expression of MEK-1 in gastric cancer cells metastasized to lymph nodes was higher than that of the primary site. CONCLUSION: MAPKs, particularly ERK subclass are overexpressed in the majority of gastric cancers. Overexpression of ERKs is correlated to TNM staging, serosa invasion, and lymph node involvement. The overexpression of p38 most likely plays a prominent role in certain morphological subtypes of gastric cancers. MEK-1 is also overexpressed in gastric cancer, particularly in metastatic lymph nodes. Upregulation of MAPK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.     

High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis.

Exposure of human ovarian tumor cell lines to cisplatin led to development of cell lines that exhibited increasing degrees of drug resistance, which were closely correlated with increase of the levels of cellular glutathione. Cell lines were obtained that showed 30- to 1000-fold increases in resistance; these cells also had strikingly increased (13- to 50-fold) levels of glutathione as compared with the drug-sensitive cells of origin. These levels of resistance to cisplatin and the cellular glutathione levels are substantially greater than previously reported. Very high cisplatin resistance was associated with enhanced expression of mRNAs for gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase; immunoblots showed increase of gamma-glutamylcysteine synthetase but not of glutathione synthetase. Glutathione S-transferase activity was unaffected, as determined with chlorodinitrobenzene as a substrate. These studies suggest the potential value of examining regulation of glutathione synthesis as an indicator of clinical prognosis. The highly resistant cell lines are proving useful for studying the multiple mechanisms by which tumor cells acquire drug- and radiation-resistance.