RKIP downregulates B-Raf kinase activity in melanoma cancer cells.

The Raf-MEK-ERK protein kinase cascade is a highly conserved signaling pathway that is pivotal in relaying environmental cues from the cell surface to the nucleus. Three Raf isoforms, which share great sequence and structure similarities, have been identified in mammalian cells. We have previously identified Raf kinase inhibitor protein (RKIP) as a negative regulator of the Raf-MEK-ERK signaling pathway by specifically binding to the Raf-1 isoform. We show here that RKIP also antagonizes kinase activity of the B-Raf isoform. Yeast two-hybrid and coimmunoprecipitation experiments indicated that RKIP specifically interacted with B-Raf. Ectopic expression of RKIP antagonized the kinase activity of B-Raf. We showed that the effects of RKIP on B-Raf functions were independent of its known inhibitory action on Raf-1. The expression levels of RKIP in melanoma cancer cell lines are low relative to primary melanocytes. Forced expression of RKIP partially reverted the oncogenic B-Raf kinase-transformed melanoma cancer cell line SK-Mel-28. The low expression of RKIP and its antagonistic action on B-Raf suggests that RKIP may play an important role in melanoma turmorgenesis.

Raf-1 kinase inhibitor protein: structure, function, regulation of cell signaling, and pivotal role in apoptosis.

The acquisition of resistance to conventional therapies such as radiation and chemotherapeutic drugs remains the major obstacle in the successful treatment of cancer patients. Tumor cells acquire resistance to apoptotic stimuli and it has been demonstrated that conventional therapies exert their cytotoxic activities primarily by inducing apoptosis in the cells. Resistance to radiation and chemotherapeutic drugs has led to the development of immunotherapy and gene therapy approaches with the intent of overcoming resistance to drugs and radiation as well as enhancing the specificity to eliminate tumor cells. However, cytotoxic lymphocytes primarily kill by apoptosis and, therefore, drug-resistant tumor cells may also be cross-resistant to immunotherapy. To evade apoptosis, tumor cells have adopted various mechanisms that interfere with the apoptotic signaling pathways and promote constitutive activation of cellular proliferation and survival pathways. Thus, modifications of the antiapoptotic genes in cancer cells are warranted for the effectiveness of conventional therapies as well as novel immunotherapeutic approaches. Such modifications will avert the resistant phenotype of the tumor cells and will render them susceptible to apoptosis. Current studies, both in vitro and preclinically in vivo, have been aimed at the modification and regulation of expression of apoptosis-related gene products and their activities. A novel protein designated Raf-1 kinase inhibitor protein (RKIP) has been partially characterized. RKIP is a member of the phosphatidylethanolamine-binding protein family. RKIP has been shown to disrupt the Raf-1-MEK1/2 [mitogen-activated protein kinase-ERK (extracellular signal-regulated kinase) kinase-1/2]-ERK1/2 and NF-kappaB signaling pathways, via physical interaction with Raf-1-MEK1/2 and NF-kappaB-inducing kinase or transforming growth factor beta-activated kinase-1, respectively, thereby abrogating the survival and antiapoptotic properties of these signaling pathways. In addition, RKIP has been shown to act as a signal modifier that enhances receptor signaling by inhibiting G protein-coupled receptor kinase-2. By regulating cell signaling, growth, and survival through its expression and activity, RKIP is considered to play a pivotal role in cancer, regulating apoptosis induced by drugs or immune-mediated stimuli. Overexpression of RKIP sensitizes tumor cells to chemotherapeutic drug-induced apoptosis. Also, induction of RKIP by drugs or anti-receptor antibodies sensitizes cancer cells to drug-induced apoptosis. In this review, we discuss the discovery, structure, function, and significance of RKIP in cancer.

Prostatic androgen repressed message-1 (PARM-1) may play a role in prostatic cell immortalisation.

BACKGROUND: Prostatic androgen-repressed message-1 (PARM-1) has been cloned from the prostate. The transcript of the PARM-1 gene is overexpressed during regression of the prostate after androgen withdrawal. The regulation of PARM-1 by androgens is limited to this organ. We have studied the effects of PARM-1 overexpression in malignant prostate cells. METHODS: The PARM-1 cDNA was introduced into the rat cancer cell line MAT LyLu along with a doxycycline-dependent regulator. RESULTS: Maximal expression of PARM-1 (fivefold induction) was achieved by incubating the cells with 2 microM doxycycline for 48 hr. A study investigating the effect of PARM-1 overexpression on the transcription of 588 genes has shown that the TLP1 gene (encoding rat telomerase protein component 1) was the most up-regulated (fourfold). In addition, a dose-dependent increase in telomerase activity was observed in cells overexpressing PARM-1. In vivo, the androgen-deprived prostate showed an increased TLP1 level and increased telomerase activity. CONCLUSIONS: Increased telomerase activity is often associated with the immortalisation of cancer cell lines, particularly prostatic ones. This could mean that PARM-1 is involved, via increased telomerase activity, in a survival program enabling certain prostatic cells to resist apoptosis, thus conferring a selective advantage to pre-cancerous or cancerous cells.

Effect of prostatein, the major protein produced by the rat ventral prostate, on phagocytic cell functions.

PROBLEM: To determine whether prostatein, the major protein produced and secreted into the seminal fluid by the rat ventral prostate has any effect on the phagocytic cell functions in vitro. METHOD OF STUDY: Analysis was done by determining if purified prostatein added to cells obtained from the peritoneal cavity has any effect on their phagocytic and intracellular killing capacity. Also, we analyzed the effect of prostatein on the production of oxygen and nitrogen intermediates, measuring these metabolites by Nitroblue tetrazolium assay and by the Griess reaction respectively. RESULTS: Prostatein possess the ability to inhibit in vitro the phagocytic and killing properties of peritoneal rat leukocytes in a dose-dependent manner. The addition of a polyclonal antiserum against prostatein specifically blocks this inhibitory effect. Moreover, prostatein inhibits the production of oxygen and nitrogen intermediates by these cells. CONCLUSION: Regulation of the production of reactive oxygen species in the reproductive tract is extremely necessary to avoid their deleterious effects on the sperm motility and the fertilization process. We propose that prostatein, a protein supplied by an accessory gland like prostate, can inhibit the macrophage function, showing an important antioxidant effect.

Identification of rat prostatic steroid binding protein (PSBP) as an immunosuppressive factor.

Prostatic steroid binding protein (PSBP) is the major protein produced ( approximately 20% of the total cytosolic protein) and secreted into the seminal fluid by the rat ventral prostate but its physiological function has not been elucidated yet. Since PSBP is secreted into the seminal fluid (which is itself a potent immunosuppressor) and has strong homology with uteroglobin (which possess an important anti-inflammatory function) our aim was to determine what effect, if any, PSBP would have on the immune system. With that purpose in mind we performed mononuclear cell cultures in the presence or absence of purified PSBP and analysed the effect of this protein on different functional parameters. PSBP inhibits the mitogen-induced proliferation of normal rat spleen mononuclear cells (MNC) specifically and in a dose-dependent manner. It reduces the production of IL-2 and the expression of its receptor (analysed by flow cytometry) which are important events for lymphocyte proliferation. Also, PSBP was able to inhibit OVA-specific proliferation of lymph node cells from previously primed animals. The immunosuppressive effect of PSBP is not due to an inherent toxic effect to the cells, since the cell viability was kept intact at the different times of culture studied. We also analysed the effect of rat PSBP on mitogen-induced proliferation of mouse spleen and human blood MNC. The proliferation was strongly abolished in a dose-dependent and non-species specific fashion. Moreover, PSBP strongly inhibits the human mixed lymphocyte reaction. Taken together, the present data support evidence for a new type of function for PSBP. We report that PSBP is a potent immunosuppressor factor and we describe its effect on the immune function in vitro. Here, we discuss the possible implications of these findings in the protection of sperm from immunologic damage in the feminine reproductive tract.

Growth response of adult germ cells to rat androgen-binding protein and human sex hormone-binding globulin.

Androgen-binding protein (ABP) is produced by Sertoli cells depending on the development and the stage of the spermatogenic cycle. Germ cell proliferation is at its peak when ABP is at its peak and secreted towards the testicular basal compartment containing spermatogonia and premeiotic spermatocytes. Rat isolated adult germ cell DNA synthesis was studied in vitro in the presence of ABP with and without steroids and in the presence of pure or recombinant sex steroid hormone-binding globulin (SHBG) using thymidine incorporation. Results are: SHBG is able to promote DNA synthesis in the absence of cofactors. Testosterone reacted negatively to the stimulatory effect of SHBG. We conclude that ABP, the physiological steroid-binding protein, should be considered as a paracrine regulator of spermatogenic DNA synthesis in the adult rat.

Intraluminal androgen binding protein alters 3H-androgen uptake by rat epididymal tubules in vitro.

Previous experiments have shown that androgen binding protein (ABP) and androgens exist in high concentrations in the tissue and the lumen of the rat caput epididymis. The present experiments were performed to determine whether or not intraluminal APB affects tubule net uptake of androgens. Caput epididymal tubules were dissected into 2-cm segments, subjected to microperfusion into the tubule lumen, and incubated for 2.5 h in 35 degrees C minimum essential medium (MEM) containing 2.0 ng tritiated testosterone (3H-T) per ml. 14C-polytheylene glycol [PEG] was included as a contamination marker. In the first series of experiments, caput tubules were perfused with a control, artificial perfusion (MKB) containing no ABP or fresh rat rete testis fluid (RTF), which is known to contain ABP. Tubules incubated while containing RTF took up 138% of the tritiated androgens taken up by control tubules. In the second series of experiments, tubules were perfused with fresh caput epididymal lumen content, MKB alone, MKB containing either 5.0 ng purified rat ABP/microliters or 50 ng ABP/microliters. Tubules incubated while containing perfused MKB took up only 47% of the tritiated androgens taken up by tubules containing perfused native lumen content. Increasing intraluminal ABP concentrations in the MKB medium increased 3H-androgen uptake in a stepwise fashion. Intraluminal ABP at a concentration of 50 ng/microliters was associated with a 71% return of 3H-androgen uptake towards that amount of 3H-androgen taken up by tubules perfused with native lumen content. Intraluminal ABP enhances net androgen uptake by caput epididymal tubules from their surrounding medium in vitro.

The androgen-dependent rat prostate protein, probasin, is a heparin-binding protein that co-purifies with heparin-binding growth factor-1.

Rat prostate extracts contain an abundant 20-22 kilodalton heparin-binding protein with near identical chromatographic properties, but only 0.2-1% of the mitogenic activity, of bovine brain heparin-binding growth factor-1 (acidic fibroblast growth factor). Amino terminal amino acid sequence (met-met-thr-asp-lys-asn-leu-lys-lys-lys-ile-glu-gly-asn-trp-arg-thr-val -tyr- leu-ala-ala-ser-?-val-glu-lys-ile-asn-glu-gly-ser-pro) and immunochemical analysis revealed that the protein is identical to the androgen-dependent protein "probasin".

[Paracrine regulation of testicular function]. / Régulation paracrine des fonctions testiculaires.

Spermatogenesis and spermiogenesis are controlled by FSH and testosterone but need also the participation of several paracrine and autocrine mechanisms of regulations. The relationships between peritubular, Sertoli and Leydig cells are currently investigated. High intratesticular testosterone levels are maintained by a binding to a protein called ABP which is synthetized by Sertoli cell and regulated by pituitary FSH. Leydig cell testosterone, peritubular cell P-Mod-S (protein modulating Sertoli function) and Sertoli cell FRP (follicle regulatory protein). Accumulation of testosterone results to aromatase activity modulation. Aromatization is stimulated by FSH, activin, alpha-MSH but is inhibited by aromatase inhibitor, inhibin, FSHBI (FSH binding inhibitor). Other molecules, growing factors, mitogenic factors, energetic substrates are synthetized in the testis under the control of germ cells. Understanding of these mechanisms of intratesticular regulation will permit to discover therapies capable of correcting certain fertility dysfunctions.

Regulation of prostatic steroid binding protein mRNAs by testosterone.

Testosterone regulates the expression of prostatic steroid binding protein by stimulating the accumulation of C1, C2 and C3 mRNA. We have used intervening sequence RNA probes to quantitate the primary transcript and a processed intermediate for C1 and C3 in nuclear RNA. Both C1 and C3 primary transcript concentration declined by about 100-fold 3 days after castration and were induced by testosterone within 1 h. In view of the magnitude and kinetics of the response we conclude that testosterone acts primarily within the cell nucleus to increase steady-state levels of nuclear RNA.