Effects of galectin-1 and galectin-3 expression on prostate cancer cell adhesion, differentiation, proliferation, and tumorigenicity

The vertebrate $\beta$-galactoside-binding lectins galectin-1 and galectin-3 have been proposed to function in diverse cellular processes such as adhesion, proliferation, differentiation, and tumorigenesis. Experiments were initiated to further study the functional properties of these molecules. A prostate cancer cell line, LNCaP, was identified which expressed neither galectin. This line was stably transfected with cDNA for either galectin-1 or galectin-3. The resultant clones were used to study effects on critical cell processes. LNCaP cells expressing galectin-1 on the surface were found to bind more rapidly than control lines to the human extracellular matrix proteins laminin and fibronectin, although overall binding was not increased. To analyze effects on differentiation, LNCaP cells were studied which had either been transfected with galectin-1 or which had been induced to express endogenous galectin-1 by treatment with the differentiation agent sodium butyrate. In both cases, cells displayed a slower rate of growth and increased rate of apoptosis. A transient decrease in expression of prostate specific antigen was seen in the butyrate treated cells but not in the transfected cells. To investigate the role of galectins in the process of malignant transformation and progression, immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded sections of human prostate tissue, the premalignant lesion prostatic intraepithelial neoplasia, primary adenocarcinoma of the prostate, and foci of metastatic prostate cancer. Galectin-1 expression was relatively constant throughout in contrast to galectin-3 which demonstrated significantly less expression in primary and metastatic tumors. LNCaP cells transfected with galectin-3 cDNA displayed lower proliferation rates, increased spontaneous apoptosis, and G1 growth phase arrest compared to controls. Four of six galectin-3 lines tested were less tumorigenic in nude mice than controls. The following conclusions are drawn regarding the role of galectin-1 and galectin-3 expression in the context of prostate cancer: (1) galectin-1 may participate in the early stages of cancer cell adhesion to extracellular matrix proteins; (2) galectin-1 expression results in a differentiated phenotype and may contribute to differentiation induction by butyrate; (3) galectin-3 expression correlates inversely with prostate cell tumorigenesis and prostate cancer metastasis. ^

Antitumor activity of an Ets protein, PEA3, in breast cancer cell lines MDA-MB-361DYT2 and BT474M1

Polyomavirus enhancer activator 3 (PEA3) is a member of the Ets family of transcription factors. We demonstrated in a previous study that, through down-regulating the HER-2/neu oncogene at the transcriptional level, PEA3 can inhibit the growth and tumor development of HER-2/neu-overexpressing ovarian cancer cells. Here, we established stable clones of the human breast cancer cell line MDA-MB-361DYT2 that express PEA3 under the control of a tetracycline-inducible promoter. The expression of PEA3 in this cell line inhibited cell growth and resulted in cell cycle delay in the G1 phase independently of the HER-2/neu down-regulation. In an orthotopic breast cancer model, we showed that expression of PEA3 inhibited tumor growth and prolonged the survival of tumor-bearing mice. In a parallel experiment in another breast cancer cell line, BT474M1, we were unable to obtain stable PEA3-inducible transfectants, which suggests that PEA3 possessed a strong growth inhibitory effect in this cell line. Indeed, PEA3 coupled with the liposome SN2 demonstrated therapeutic effects in mice bearing tumors induced by BT474M1. These results provide evidence that the PEA3 gene could function as an antitumor and gene therapy agent for human breast cancers. ^

The characterization of PKC-isozyme specific EGFR-dependent Erk1/2 activation in androgen-independent prostate cancer cell lines

One growth factor receptor commonly altered during prostate tumor progression is the epidermal growth factor receptor (EGFR). EGFR signaling regulates Erk1/2 phosphorylation through multiple mechanisms. We hypothesized that PKC isozymes play a role in EGFR-dependent signaling, and that through PKC isozyme selective inhibition, EGFR-dependent Erk1/2 activation can be attenuated in AICaP cells. ^ To test the hypothesis, PKC activation was induced by 12-O-tetradecanoyi-phorbol-13-acetate (TPA) in PC-3 cells. As a result, Erk1/2 was activated similarly to what was observed upon EGF stimulation. EGF-induced Erk1/2 activation in PC-3 cells was PKC-dependent, as demonstrated through use of a selective PKC inhibitor, GF109203X. This provides evidence for PKC regulatory control over Erk1/2 signaling downstream of EGFR. Next, we demonstrated that when PKC was inhibited by GF109203X, EGF-stimulated Erk1/2 activation was inhibited in PC-3, but not DU145 cells. TPA-stimulated Erk1/2 activation was EGFR-dependent in both DU145 and PC-3 cells, demonstrated through abrogation of Erk1/2 activation by a selective EGFR inhibitor AG1478. These data support PKC control at or upstream of EGFR in AICaP cells. We observed that interfering with ligand/EGFR binding abrogated Erk1/2 signaling in TPA-stimulated cells, revealing a role for PKC upstream of EGFR. ^ Next, we determined which PKC isozymes might be responsible for Erk1/2 regulation. We first determined that human AICaP cell lines express the same PKC isozymes as those observed in clinical prostate cancer specimens (α, ϵ, &zgr;, ι and PKD). Isozyme-selective methods were employed to characterize discrete PKC isozyme function in EGFR-dependent Erk1/2 activation. Pharmacologic inhibitors implicated PKCα in TPA-induced EGFR-dependent Erk1/2 activation in both PC-3 and DU145 cells. Further, the cPKC-specific inhibitor, Gö6976 decreased viablilty of DU145 cells, providing evidence that PKCα is necessary for growth and survival. Finally, resveratrol, a phytochemical with strong cancer therapeutic potential inhibited Erk1/2 activation, and this correlated with selective inhibition of PKCα. These results demonstrate that PKC regulates pathways critical to progression of CaP cells, including those mediated by EGFR. Thus, PKC isozyme-selective targeting is an attractive therapeutic strategy, and understanding the role of specific PKC isozymes in CaP cell growth and survival may aid in development of effective, non-toxic PKC-targeted therapies. ^

XIPHOPHORUS ENZYME GENE EXPRESSION: TISSUE SPECIFICITY, GENETIC CONTROL, AND STABILITY IN CULTURED CELLS

Five permanent cell lines were developed from Xiphophorus maculatus, X. helleri, and their hybrids using three tissue sources, including adults and embryos of different stages. To evaluate cell line gene expression for retention of either tissue-of-origin-specific or ontogenetic stage-specific characters, the activity distribution of 44 enzyme loci was determined in 11 X. maculatus tissues, and the developmental genetics of 17 enzyme loci was charted in X. helleri and in helleri x maculatus hybrids using starch gel electrophoresis. In the process, eight new loci were discovered and characterized for Xiphophorus.^ No Xiphophorus cell line showed retention of tissue-of-origin-specific or ontogenetic stage-specific enzyme gene expressional traits. Instead, gene expression was similar among the cell lines. One enzyme, adenosine deaminase (ADA) was an exception. Two adult-origin cell lines expressed ADA, whereas, three cell lines derived independently from embryos did not. ADA('-) expression of Xiphophorus embryo-derived cell lines may represent retention of an embryonic gene expressional trait. In one cell line (T(,3)) derived from 13 pooled interspecific hybrid (F(,2)) embryos, shifts with time were observed at enzyme loci polymorphic between the two species. This suggested shifts in ratios of cells of different genotypes in the population rather than unstable gene expression in one dominant cell type.^ Verification of this hypothesis was attempted by cloning the culture--seeking clones having different genetic signatures. The large number of loci electrophoretically polymorphic between the two species and whose alleles segregated independently into the 13 progeny from which this culture originated almost guaranteed the presence of different genetic signatures (lineages) in T(,3).^ Seven lineages of cells were found within T(,3), each expressing genotypes at some loci not characteristic of the expression of the culture-as-a-whole, supporting the hypothesis tested. Quantitative studies of ADA expression in the whole culture (ADA('-)) and in clones of these seven lineages suggested the predominance in T(,3) of ADA deficient cell lineages, although moderate to high ADA output clones also occurred. Thus, T(,3) has the potential to shift phenotypes from ADA('-) to ADA('+) by simply changing proportions of its constituent cell types, demonstrating that such shifts can occur in any cell culture containing cells of mixed expressional characteristics.^

Regulation of phosphatidylinositide turnover in the myometrium by cAMP: Implications for the control of uterine contraction

Regulation of uterine quiescence involves the integration of the signaling pathways regulating uterine contraction and relaxation. Uterine contractants increase intracellular calcium through receptor/GαqPLC coupling, resulting in contraction of the myometrium. Elevation of cAMP concentration has been correlated with relaxation of the myometrium. However, the mechanism of cAMP action in the uterus is unclear. ^ Both endogenous and exogenous increases in cAMP inhibited oxytocin-stimulated phosphatidylinositide turnover in an immortalized pregnant human myometrial cell line (PHM1-41). This inhibition was reversed by cAMP-dependent protein kinase (PKA) inhibitors, suggesting the involvement of PKA. cAMP inhibited phosphatidyinositide turnover stimulated by different agonists in different cell lines. These data suggest that the cAMP inhibitory mechanism is neither cell nor receptor dependent, and inhibits Gαq/PLCβ1 and PLCβ3 coupling. ^ The subcellular localization of PKA occurs via PKA binding to A-Kinase-Anchoring-Proteins (AKAP), and peptides that inhibit this association have been developed (S-Ht31). S-Ht31 blocked cAMP-stimulated PKA activity and decreased PKA concentration in PHM1-41 cell plasma membranes. S-Ht31 reversed the ability of CPT-cAMP, forskolin and relaxin to inhibit phosphatidylinositide turnover in PHM1-41 cells. Overlay analysis of both PHM1-41 cell and nonpregnant rat myometrium found an AKAPs of 86 kDa and 150 kDa associated with the plasma membrane, respectively. These data suggest that PKA anchored to the plasma membrane via AKAP150/PKA anchoring is involved in the cAMP inhibitory mechanism. ^ CPT-cAMP and isoproterenol inhibited phosphatidylinositide turnover in rat myometrium from days 12 through 20 of gestation. In contrast, neither agent was effective in the 21 day pregnant rat myometrium. The decrease in the cAMP inhibitory mechanism was correlated with a decrease in PKA and an increase in protein phosphatase 2B (PP2B) concentration in rat myometrial plasma membranes on day 21 of gestation. In myometrial total cell homogenates, both PKA and PP2B concentration increased on day 21. S-Ht31 inhibited cAMP inhibition of phosphatidylinositide turnover in day 19 pregnant rat myometrium. Both PKA and PP2B coimmunoprecipitated with an AKAP150 in a gestational dependent manner, suggesting this AKAP localizes PKA and PP2B to the plasma membrane. ^ These data presented demonstrate the importance of the cAMP inhibitory mechanism in regulating uterine contractility. ^