Bax-alpha promotes apoptosis induced by cancer chemotherapy and accelerates the activation of caspase 3-like cysteine proteases in p53 double mutant B lymphoma Namalwa cells

Stable transfected human p53 (mt/mt) B lymphoma Namalwa variant lines showing differential expression of the Bax-alpha protein were derived under hygromycin selection. Overexpression of Bax-alpha in these variant cells accelerates cell death induced by short or continuous treatments with various concentrations of camptothecin, etoposide, vinblastine and shows no accelerating cell death activity in cis-platinum and paclitaxel-treated cells. Activation of apoptosis and oligonucleosome-sized DNA fragmentation was observed in the variant lines with more pronounced effect in cells containing high level of Bax-alpha protein. These results suggest that increased cell death mediated by anticancer drugs correlates with Bax-alpha level of expression and that Bax-alpha sensitizes Namalwa cells treated at low drug concentrations. The extent of DNA synthesis inhibition following DNA topoisomerase inhibitor treatments was similar in control and all transfected Namalwa cells suggesting that Bax-alpha acts downstream of DNA topoisomerase-mediated DNA strand breaks. To define further the relation between Bax-alpha expression and apoptosis activation, kinetics of caspase activation was measured in drug-treated cells. Caspase activities were measured using specific fluorogenic peptide derivatives DABCYL-YVADAPV-EDANS and Ac-DEVD-AMC, substrates of the caspase 1-like and caspase 3-like families, respectively. In control and Bax-alpha transfected Namalwa cells no increase in caspase 1-like activity was detected following camptothecin and etoposide treatments. In contrast, a significant difference in Ac-DEVD-AMC hydrolysis activity was observed in Bax-alpha transfected Namalwa cells compared to that of control Namalwa cells after camptothecin and etoposide treatment. Increased caspase 3-like activity correlated also with poly(ADPribosyl) polymerase cleavage. Taken together, these results suggest that Bax-alpha sensitize B lymphoma cells to series of anticancer drugs and accelerates the activation of apoptotic protease cascade.     

Activation of the luteinizing hormone beta promoter by gonadotropin-releasing hormone requires c-Jun NH2-terminal protein kinase

Regulation of the mitogen-activated protein kinase (MAPK) family by gonadotropin-releasing hormone (GnRH) in the gonadotrope cell line LbetaT2 was investigated. Treatment with gonadotropin-releasing hormone agonist (GnRHa) activates extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). Activation of ERK by GnRHa occurred within 5 min, and declined thereafter, whereas activation of JNK by GnRHa occurred with a different time frame, i.e. it was detectable at 5 min, reached a plateau at 30 min, and declined thereafter. GnRHa-induced ERK activation was dependent on protein kinase C or extracellular and intracellular Ca(2+), whereas GnRHa-induced JNK activation was not dependent on protein kinase C or on extracellular or intracellular Ca(2+). To determine whether a mitogen-activated protein kinase family cascade regulates rat luteinizing hormone beta (LHbeta) promoter activity, we transfected the rat LHbeta (-156 to +7)-luciferase construct into LbetaT2 cells. GnRH activated the rat LHbeta promoter activity in a time-dependent manner. Neither treatment with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD98059, nor cotransfection with a catalytically inactive form of a mitogen-activated protein kinase construct inhibited the induction of the rat LHbeta promoter by GnRH. Furthermore, cotransfection with a dominant negative Ets had no effect on the response of the rat LHbeta promoter to GnRH. On the other hand, cotransfection with either dominant negative JNK or dominant negative c-Jun significantly inhibited the induction of the rat LHbeta promoter by GnRH. In addition, GnRH did not induce either the rat LHbeta promoter activity in LbetaT2 cells transfected stably with dominant negative c-Jun. These results suggest that GnRHa differentially activates ERK and JNK, and a JNK cascade is necessary to elicit the rat LHbeta promoter activity in a c-Jun-dependent mechanism in LbetaT2 cells.     

Prolactin-releasing peptide activation of the prolactin promoter is differentially mediated by extracellular signal-regulated protein kinase and c-Jun N-terminal protein kinase

Regulation of the mitogen-activated protein kinase (MAPK) family by prolactin-releasing peptide (PrRP) in both GH3 rat pituitary tumor cells and primary cultures of rat anterior pituitary cells was investigated. PrRP rapidly and transiently activated extracellular signal-regulated protein kinase (ERK) in both types of cells. Both pertussis toxin, which inactivates G(i)/G(o) proteins, and exogenous expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, completely blocked the PrRP-induced ERK activation, suggesting the involvement of G(i)/G(o) proteins in the PrRP-induced ERK activation. Down-regulation of cellular protein kinase C did not significantly inhibit the PrRP-induced ERK activation, suggesting that a protein kinase C-independent pathway is mainly involved. PrRP-induced ERK activation was not dependent on either extracellular Ca(2+) or intracellular Ca(2+). However, the ERK cascade was not the only route by which PrRP communicated with the nucleus. JNK was also shown to be significantly activated in response to PrRP. JNK activation in response to PrRP was slower than ERK activation. Moreover, to determine whether a MAPK family cascade regulates rat prolactin (rPRL) promoter activity, we transfected the intact rPRL promoter ligated to the firefly luciferase reporter gene into GH3 cells. PrRP activated the rPRL promoter activity in a time-dependent manner. Co-transfection with a catalytically inactive form of a MAPK construct or a dominant negative JNK, partially but significantly inhibited the induction of the rPRL promoter by PrRP. Furthermore, co-transfection with a dominant negative Ets completely abolished the response of the rPRL promoter to PrRP. These results suggest that PrRP differentially activates ERK and JNK, and both cascades are necessary to elicit rPRL promoter activity in an Ets-dependent mechanism.     

Opposing regulation of B cell receptor-induced activation of mitogen-activated protein kinases by CD45

In this study, we examined the contribution made by CD45 to B cell antigen receptor (BCR)-induced activation of mitogen-activated protein kinase (MAPK) family members. We found that CD45 negatively regulated BCR-induced c-Jun NH(2)-terminal kinase (JNK) and p38 activation in immature WEHI-231 cells, whereas in mature BAL-17 cells, CD45 positively regulated JNK and p38 activation and negatively regulated extracellular signal-regulated kinase activity. Furthermore, cooperative action of JNK and p38 dictated BCR-induced inhibition of growth. Thus, CD45 appears to differentially regulate BCR-induced activation of MAPK members, and can exert opposing effects on JNK and p38 in different cellular milieu, controlling the B cell fate.     

Different sensitivity to interleukin 4 of interleukin 2- and interferon alpha-induced CD69 antigen expression in human resting NK cells and CD3+, CD4-, CD8- lymphocytes.

The effect of rIL-4 on CD69 antigen expression induced by rIL-2 or by rINF-alpha on human resting NK cells and CD3+, CD4-, CD8- T lymphocytes has been investigated. rIL-4 drastically inhibited CD69 antigen expression induced by rIL-2 in both cell types. In contrast, rIL-4 did not alter rINF-alpha-induced CD69 antigen expression. Consistent results were obtained evaluating the cytolytic activity of NK cells against the Raji target cell line: rINF-alpha-induced lytic activity was not inhibited by rIL-4, while rIL-2-induced lytic activity was drastically inhibited. Proliferative activity of NK cells induced by rIL-2, in contrast, was only slightly reduced by rIL-4. rIL-4 did not alter the expression of the beta chain of IL-2 receptor, evaluated in NK cells by indirect immunofluorescence. Expression of the alpha chain of IL-2 receptor could not be detected in NK cells by indirect immunofluorescence. It can therefore be suggested that the selective inhibitory effect of rIL-4 on rIL-2-induced activation of NK cells is not mediated by downregulation of alpha and beta chains of IL-2 receptor.     

Cloning and functional expression of the T cell activation antigen CD26.

A cDNA encoding the T cell activation Ag CD26 was isolated from human PHA-activated T cells by using an expression cloning method. The nucleotide sequence obtained predicts a protein of 766 amino acids of type II membrane topology, with six amino acids in the cytoplasmic region. The predicted amino acid sequence of the Ag was 85% homologous to that of the dipeptidyl peptidase IV enzyme isolated from rat liver. Derivatives of the human leukemic T cell line Jurkat transfected with a CD26 expression plasmid were established. Characterization of the CD26 Ag expressed by the transfected Jurkat cells revealed that the Ag could be immunoprecipitated as a 110-kDa molecule similar to that found on peripheral blood T cells and that the Ag had dipeptidyl peptidase IV activity. Functional analysis of these Jurkat transfectants showed that cross-linking of the CD26 and CD3 Ag with their respective antibodies resulted in enhanced intracellular calcium mobilization and IL-2 production. These results provide direct evidence that the CD26 Ag plays a role in T cell activation.     

Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein.

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.     

Reversing chemoresistance in cisplatin-resistant human ovarian cancer cells: a role of c-Jun NH2-terminal kinase 1

To investigate the role of activation of c-Jun NH2-terminal kinase 1 (JNK1) in mediating cisplatin-induced apoptosis and the possibility of induction of JNK activity in triggering relation to DNA damage and drug resistance. We investigated the difference of cisplatin-induced activation of JNK pathway and H2O2 alteration between cisplatin-sensitive human ovarian carcinoma cell line A2780 and its resistant variant A2780/DDP. JNK, p-JNK protein, and extracellular H2O2 levels were determined in both A2780 and A2780/DDP cells which were transfected with dominant negative allele of JNK and recombinant JNK1 separately. Both A2780 and A2780/DDP were treated with CDDP, the JNK pathway was activated and a prolonged JNK activation was maintained for at least 12 h in A2780, and only a transient activation (3 h) was detected in A2780/DDP in response to cisplatin treatment. Inhibition of JNK activity by transfection with a dominant negative allele of JNK blocked CDDP-induced apoptosis significantly in A2780 cells. Selective stimulation of the JNK pathway by lipofectamine-mediated delivery of recombinant JNK1 led to activation of c-Jun and decrease of extracellular H2O2, as well as apoptosis sensitization to CDDP in A2780/DDP cells. We concluded that JNK pathway might play an important role in mediating cisplatin-induced apoptosis in A2780 cells, and the duration of JNK activation might be critical in determining whether cells survive or undergo apoptosis. The resistance to CDDP can be reversed through activating c-Jun and decreasing extracellular generation of H2O2 by pcDNA3(FLAG)-JNK1-wt transfection in A2780/DDP cells.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

转化生长因子β激活性激酶在胶质细胞信号转导中的作用机制

丝裂原激活蛋白激酶（MAPK）和 NFκB介导了炎症细胞转录活性的信号转导过程.转化生长因子β激活性激酶（TGFβ-activated kinase 1,TAK1）是这些转导通路的上游激酶.通过在胶质细胞株中瞬时转染TAK1和它的结合蛋白因子（TAK1-binding protein1 TAB1）基因,或与iNOS(可诱导型氧化氮合酶基因)启动子报告基因（iNOS-Luc）质粒共转染,探讨中枢两类胶质细胞在炎症反应过程中TAK1诱导iNOS 和细胞因子表达的作用机制.结果显示,TAK1明显激活iNOS 和细胞因子（TNFα、IL-1、IL-6）的表达活性. 而且当使用它的下游激酶p38 MAPK、JNK和NFκB的抑制剂（SB203580、SP620125和CAPE）后,这些表达活性明显被抑制.用IκBα的磷酸化突变体质粒(IκBαM)共转染胶质细胞株,能完全抑制iNOS的表达活性.研究结果提示：在胶质细胞内的p38 MAPK、JNK和NFκB信号介导的iNOS和细胞因子的转录表达过程中,TAK1起着非常重要的调节作用.     

Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.     

重组人p53腺病毒转染淋巴瘤源性树突状细胞的抗肿瘤免疫效应

目的：研究重组人p53腺病毒转染淋巴瘤源性树突状细胞的抗肿瘤免疫效应。方法：采集本科室初诊的弥漫性大B细胞淋巴瘤（DLBCL）肿大淋巴结分离单个核细胞（MNC）进行体外DC的诱导培养,分为实验组A（rAd-p53-DC）、对照组A（rAd-DC）、空白对照组A（N-DC）,同时采集患者的外周血分离单个核细胞（MNC）,进行体外DC的诱导培养,分为实验组B（rAd-p53-DC）、对照组B（rAd-DC）、空白对照组B（N-DC）,用重组人p53腺病毒（rAd-p53）转染2种来源的DCS,流式检测DCS免疫表型,用Western-blotting鉴定P53蛋白的表达,ELISA法检测上清中的细胞因子IL-12的含量,混合淋巴细胞反应（mixed lymphocyte reac-tion,MLR）测定DCS刺激同种异体淋巴细胞增殖能力,用乳酸脱氢酶（lactate dehydrogenase,LDH）释放法检测经rAd-p53转染的两种来源DCS的细胞毒性T淋巴细胞反应（CTL）。结果：DC的表型（CD1a除外）CD83、CD80、CD86和HLA-DR实验组均较对照组及空白对照组明显增高（p〈0.05）。Western-blotting可检测到实验组P53蛋白的表达。上清液中IL-12分泌水平实验组均较对照组及空白对照组明显增高（p〈0.05）。实验组具有明显的刺激自体淋巴细胞增殖的能力,且刺激能力随rAd-p53-DC与淋巴细胞比例的增加而升高。对照组及空白对照组也能刺激同种自体淋巴细胞增殖的能力,但较实验组差（p〈0.05）。对靶细胞的细胞毒作用（CTL效应）结果显示,实验组介导同种异体的淋巴细胞杀伤率显著高于对照组及空白对照组（P〈0.05）。且实验组A的CTL效应明显高于实验组B,两者之间有显著性差异（P〈0.05）。结论：rAd-p53-DC为基础的肿瘤疫苗有可能在解决淋巴瘤的MRD、DC免疫耐受等问题上发挥强大的治疗作用。     

Positive signaling through CD72 induces mitogen-activated protein kinase activation and synergizes with B cell receptor signals to induce X-linked immunodeficiency B cell proliferation

CD72 is a 45-kDa B cell transmembrane glycoprotein that has been shown to be important for B cell activation. However, whether CD72 ligation induces B cell activation by delivering positive signals or sequestering negative signals away from B cell receptor (BCR) signals remains unclear. Here, by comparing the late signaling events associated with the mitogen-activated protein kinase pathway, we identified many similarities and some differences between CD72 and BCR signaling. Thus, CD72 and BCR activated the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinase. Both CD72- and BCR-mediated ERK and JNK activation required protein kinase C activity, which was equally important for CD72- and BCR-induced B cell proliferation. However, CD72 induced stronger JNK activation compared with BCR. Surprisingly, the JNK activation induced by both BCR and CD72 is Btk independent. Although both CD72 and BCR induced Btk-dependent ERK activation, CD72-mediated proliferation is more resistant to blocking of ERK activity than that of BCR, as shown by the proliferation response of B cells treated with PD98059 and dibutyryl cAMP, agents that inhibit ERK activity. Most importantly, CD72 signaling compensated for defective BCR signaling in X-linked immunodeficiency B cells and partially restored the proliferation response of X-linked immunodeficiency B cells to anti-IgM ligation. These results suggest that CD72 signals B cells by inducing BCR-independent positive signaling pathways.     

Involvement of the JNK activation in terbinafine‐induced p21 up‐regulation and DNA synthesis inhibition in human vascular endothelial cells

Previously, we demonstrated that the extracellular signal‐regulated kinase (ERK)‐mediated pathway contributes to the terbinafine (TB)‐induced increases of p21 and p53 protein level as well as decrease of DNA synthesis in human umbilical venous endothelial cells (HUVEC). The aim of this study is to examine the involvement of c‐Jun NH2‐terminal kinase (JNK) in the TB‐induced increase of p21 protein level and DNA synthesis inhibition. Western blot analysis and kinase assay demonstrated that TB treatment increased both the protein level and the kinase activity of JNK1/2 in HUVEC. Transfection of HUVEC with JNK1 dominant negative (DN‐JNK1) prevented the TB‐induced increases of p21 and p53 protein level and decrease of DNA synthesis, suggesting that JNK1/2 activation is involved in the TB‐induced cell cycle arrest in HUVEC. Moreover, over‐expression of mitogen‐activated protein kinase (MEK)‐1 prevented the TB‐induced increase of JNK1/2 protein levels, suggesting that MEK‐1 is an upstream inhibitor of JNK. Transfection of HUVEC with DN‐JNK1 prevented the TB‐induced inhibition of ERK phosphorylation, suggesting that JNK1/2 might serve as a negative regulator of ERK. Taken together, our results suggest that JNK activation is involved in the TB‐induced inhibition of ERK phosphorylation, p53 and p21 up‐regulation and DNA synthesis inhibition in HUVEC. J. Cell. Biochem. 108: 860–866, 2009. © 2009 Wiley‐Liss, Inc.     

CD28 is not required for c-Jun N-terminal kinase activation in T cells.

Studies in Jurkat cells have shown that combined stimulation through the TCR and CD28 is required for activation of c-Jun N-terminal kinase (JNK), suggesting that JNK activity may mediate the costimulatory function of CD28. To examine the role of JNK signaling in CD28 costimulation in normal T cells, murine T cell clones and CD28(+/+) or CD28(-/-) TCR transgenic T cells were used. Although ligation with anti-CD28 mAb augmented JNK activation in Th1 and Th2 clones stimulated with low concentrations of anti-CD3 mAb, higher concentrations of anti-CD3 mAb alone were sufficient for JNK activation even in the absence of anti-CD28. JNK activity was comparably induced in both CD28(+/+) and CD28(-/-) 2C/recombinase-activating gene 2(RAG2)(-/-) T cells stimulated with anti-CD3 mAb alone, and with L(d)/peptide dimers, a direct alphabeta TCR ligand. Moreover, JNK activation was also detected in 2C/RAG2(-/-) T cells stimulated with P815 cells that express the relevant alloantigen L(d) whether or not B7-1 was coexpressed. However, IL-2 production by both Th1 clones and CD28(+/+) 2C/RAG2(-/-) T cells was detected only upon TCR and CD28 coengagement. Thus, CD28 coligation is not necessary, and stimulation through the TCR is sufficient, for JNK activation in normal murine T cells. The concept that JNK mediates the costimulatory function of CD28 needs to be reconsidered.     

Prolactin stimulates activation of c-jun N-terminal kinase (JNK)

In recent years the mitogen-activated protein (MAP) kinase family has expanded to include both c-jun N-terminal kinases (JNKs), and the p38/HOG1 family in addition to the extracellular regulated kinase (ERK) family. These kinases are activated by a variety of growth factors, as well as extra- and intracellular insults such as osmotic stress, UV light, and chemotherapeutic agents. Stimulation of the PRL-dependent Nb2 cell line with PRL results in the rapid activation of JNK as determined by the glutathione-S-transferase (GST)-jun kinase assay. Activation was maximal 30 min after stimulation with 50 nM rat PRL (rPRL) and decreased after that time. Dose response studies indicated that concentrations as low as 10 nM rPRL resulted in maximal activation. The interleukin-3 (IL-3)-dependent myeloid progenitor cell line 32Dcl3 was transfected with the long, Nb2, and short forms of the rat PRL receptor (rPRLR), as well as the long form of the human PRLR (hPRLR). The long and Nb2 forms of the PRLR were able to stimulate activation of JNK; however, the short form of the rPRLR was not. This corresponds with the inability of the short form of the rPRLR to stimulate proliferation of 32Dcl3 cells. Activation of JNK in 32Dcl3 cells expressing the long form of the hPRLR was maximal at 30 min after stimulation with 100 nM ovine PRL (oPRL) and declined after that time. Dose response studies indicated that activation of JNK was maximal after 30 min at a concentration of 10 nM, and the amount of activated JNK declined at the highest concentration of oPRL, 100 nM. Immunoblot analysis with an antibody that recognizes the activated (phosphorylated) forms of JNK1 and JNK2 indicated that both JNK1 and JNK2 isoforms were activated in 32D/hPRLR cells stimulated with oPRL. A recombinant human adenovirus expressing a kinase-inactive mutant of JNK1 (APF mutant) was used to determine the biological effect of blocking JNK activity in Nb2 cells. Expression of the JNK1-APF mutant inhibited cellular proliferation and induced DNA fragmentation typical of cells undergoing apoptosis. These data suggest that activation of JNKs may be important in mitogenic signaling and/or suppression of apoptosis in Nb2 cells.