Differential responsiveness of prostatic acid phosphatase and prostate-specific antigen mRNA to androgen in prostate cancer cells.

Androgens regulate the expression of both human prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), two major prostate epithelium-specific differentiation antigens. Due to the important role of these two enzymes as prostate epithelium differentiation markers, we investigated their regulation of expression at the mRNA level in LNCaP human prostate carcinoma cells. Interestingly, phenol red, a pH indicator in the culture medium, promoted cell growth. To eliminate this non-specific effect, a phenol red-free, steroid-reduced medium was utilized. When high-density cells were grown in that medium, 5alpha-dihydrotestosterone (DHT) suppressed PAcP but stimulated PSA. However, tumor promoter phorbol ester 12-o-tetradecanoyl phorbol-13-acetate (TPA) functioned as a potent inhibitor of both PAcP and PSA expression. Prolonged treatment with DHT as well as TPA resulted in a similar down-regulation of protein kinase C and cellular PAcP activities. Thus, the levels of PAcP and PSA mRNA are differentially regulated by androgens in LNCaP cells.

Neuregulin in neuron/glial interactions in the central nervous system. GGF2 diminishes autoimmune demyelination, promotes oligodendrocyte progenitor expansion, and enhances remyelination.

Glial growth factor 2 (GGF2) is a neuronal signal that promotes the proliferation and survival of the oligodendrocyte, the myelinating cell of the central nervous system (CNS). This study has focused on recombinant human GGF2 (rhGGF2) and it's potential to affect clinical recovery and repair to damaged myelin in chronic relapsing experimental autoimmune encephalomyelitis (EAE) in the mouse, a major animal model for the human demyelinating disease, multiple sclerosis (MS). Mice with EAE were treated with rhGGF2 during both the acute and relapsing phases, and GGF2 treatment led to delayed signs, decreased severity and resulted in statistically significant reductions in relapse rate. Further, rhGGF2-treated groups displayed CNS lesions with more remyelination than in controls. This correlated with increased expression of myelin basic protein exon 2, a marker for remyelination, and with an increase of the regulatory cytokine, IL-10. Thus, a beneficial effect of a neurotrophic growth factor has been demonstrated upon the clinical, pathologic and molecular manifestations of autoimmune demyelination, an effect that was associated with increased expression of a Th2 cytokine. rhGGF2 treatment may represent a novel approach to the treatment of MS (Cannella et al., 1998).

The neuregulin, glial growth factor 2, diminishes autoimmune demyelination and enhances remyelination in a chronic relapsing model for multiple sclerosis.

Glial growth factor 2 (GGF2) is a neuronal signal that promotes the proliferation and survival of the oligodendrocyte, the myelinating cell of the central nervous system (CNS). The present study examined whether recombinant human GGF2 (rhGGF2) could effect clinical recovery and repair to damaged myelin in chronic relapsing experimental autoimmune encephalomyelitis (EAE) in the mouse, a major animal model for the human demyelinating disease, multiple sclerosis. Mice with EAE were treated with rhGGF2 during both the acute and relapsing phases. Clinically, GGF2 treatment delayed signs, decreased severity, and resulted in statistically significant reductions in relapse rate. rhGGF2-treated groups displayed CNS lesions with more remyelination than in controls. This correlated with increased mRNA expression of myelin basic protein exon 2, a marker for remyelination, and with an increase in the CNS of the regulatory cytokine, interleukin 10, at both the RNA and protein levels. Thus, a beneficial effect of a neurotrophic growth factor has been demonstrated on the clinical, pathologic, and molecular manifestations of autoimmune demyelination, an effect that was associated with increased expression of a T helper 2 cytokine. rhGGF2 treatment may represent a novel approach to the treatment of multiple sclerosis.

The expression of prostatic acid phosphatase is transcriptionally regulated in human prostate carcinoma cells.

The expression of prostatic acid phosphatase (PAcP) in three human prostate carcinoma cell lines including LNCaP, DU 145 and PC-3, was studied to explore its potential role as a marker in the progression of prostate cancer. Although Southern blot analysis suggested the presence of PAcP gene in all three prostate carcinoma cell lines, the Northern blot analysis and the reverse transcriptase-polymerase chain reaction (RT-PCR) assay showed that PAcP mRNA can be detected only in LNCaP cells. As one of the major differences between LNCaP cells and PC-3 as well as DU 145 cells is the androgen-sensitivity of LNCaP cells, we then focused on the influence of PAcP expression by the presence of androgen receptor (AR) in human AR cDNA-transfected PC-3 cells and high passages of LNCaP cells. The results demonstrated that the transfection of human AR cDNA into PC-3 cells did not have any detectable effect on the expression of PAcP. Further, in LNCaP cells, while the level of PAcP mRNA diminished upon passage, the AR mRNA level remained approximately the same. Together, these data suggested that the differential expression of PAcP in different prostate carcinoma cells including high passages of LNCaP cells may occur at the transcriptional level and may have little linkage to the expression of AR.

The cellular level of prostatic acid phosphatase and the growth of human prostate carcinoma cells.

Prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen. It has been demonstrated that human PAcP exhibits endogenous protein tyrosine phosphatase (PYP) activity, and that it represents the major PYP activity in normal prostate cells. Thus, it has been postulated that cellular PAcP may play a role in the tyrosine phosphorylation-mediated signal transduction. In this paper, we used LNCaP human prostate carcinoma cells, which express the endogenous PAcP, to study changes in cellular PAcP activity during cell growth. Our results demonstrated that PAcP activity increased when the cells reached confluence. Stimulation of cell growth by fresh culture medium or 5 alpha-dihydrotestosterone (DHT), a classical stimulator of prostate epithelial growth, resulted in a decline in PAcP activity. Moreover, transfection of PC-3 cells, which do not express PAcP, with a PAcP-expressing vector led to diminished cellular growth rate. These data established an inverse relationship between the cellular level of PAcP and the cell growth rate, suggesting that PAcP may be involved in regulating the growth of human prostate carcinoma cells.

Effect of cell density on androgen regulation of the mRNA level of human prostatic acid phosphatase.

The expression of human prostatic acid phosphatase (PAcP), a differentiation antigen of prostate epithelial cells, has been proposed to be regulated by androgen. We investigated this regulatory mechanism at the post-transcriptional level in LNCaP human prostate carcinoma cells, the only cultured cells that express PAcP. Our results demonstrated that the level of PAcP mRNA decreased when the cell density increased. Further, 5 alpha-dihydrotestosterone, an active form of the endogenous androgen, stimulated the PAcP mRNA level in low-density cells; while, caused a decrease in high-density cells. Thus, in LNCaP human prostate carcinoma cells, cell density could modulate PAcP expression at the mRNA level including androgen regulation.

Regulation of the expression of prostatic acid phosphatase in LNCaP human prostate carcinoma cells.

Human prostatic acid phosphatase (PAcP) is a prostate epithelium-specific differentiation antigen and its expression has been proposed to be regulated by androgens. Since cellular PAcP may function as a protein tyrosine phosphatase, we investigated the regulatory mechanism of its expression at different growth stages in LNCaP cells, the only cultured human prostate carcinoma cells that express an endogenous PAcP. Cells were plated at different densities to represent different stages of cellular growth for quantitating the expression of PAcP. In 4-d subconfluent cells, the cellular PAcP activity and protein level increased following the seeded cell density, consistent with mRNA levels. By day 7, all cultures had an approximately equal amount of total cellular proteins, indicating that cell growth approached to confluence, except the one that was plated at the lowest density. The cellular PAcP activity per cell was increased and corresponded to its protein level as observed in 4-d cultures. However, in 7-d cultured cells, although the PAcP protein level increased, its mRNA level declined. This increased PAcP protein level despite the decreased message was in part due to a prolonged half-life of the protein. Further, androgen effect on the PAcP mRNA level was also shown to be a cell density-dependent phenomenon. In low-density cultured cells, the PAcP mRNA level was elevated approximately 100% by 5 alpha-dihydrotestosterone (DHT) stimulation. However, in high-density confluent cells the mRNA level was slightly decreased by DHT treatment. Further, treatments with various growth stimulators resulted in various degrees of inhibition on PAcP mRNA levels. In conclusion, the data indicate that the cellular level of PAcP activity is associated with the cell density/confluence of LNCaP cells. Further, cell density could modulate androgen effect on PAcP expression at the mRNA level.

Regulation of prostatic acid phosphatase expression and secretion by androgen in LNCaP human prostate carcinoma cells.

The expression and the secretion of human prostatic acid phosphatase (PAcP), a differentiation antigen which is the major acid phosphatase in prostate epithelial cells, are thought to be regulated by an androgen. We investigated this regulatory mechanism at the post-transcriptional level in LNCaP human prostate carcinoma cells using a cDNA clone for the secretory form of PAcP. 5 alpha-Dihydrotestosterone (DHT, an active form of endogenous androgen) stimulated the secretion of PAcP from cells grown in a steroid-reduced medium and in a defined serum-free medium, respectively. The secreted PAcP activity was increased following a DHT dose in a dose-dependent fashion at concentrations of up to 1 microM. Further, the stimulation of PAcP secretion occurred following the period of exposure to DHT. During a 5-day treatment period, with 10 nM of DHT in the steroid-reduced medium, the secretion of PAcP was stimulated approximately 150% over that from control cells. Nevertheless, PAcP was secreted from cells grown in the absence of added DHT. First, the androgen dependency of PAcP expression was examined. The expression and the secretion of PAcP were observed in cells that were grown in a defined serum-free medium and grown in a steroid-reduced medium, in the absence of DHT. The increased secretion by DHT was further demonstrated to be in part due to an increase in PAcP mRNA level, as evidenced by Northern blot analysis. PAcP mRNA levels were elevated approximately 2-fold and corresponded to an increase of approximately 2.5-fold in the secreted level of newly synthesized 35S-PAcP. Then, the effect of DHT on the secretory process was investigated. Results of pulse-chase labeling experiments indicated that the secretory rate of PAcP was stimulated by about 50% on average by DHT. In conclusion, our data demonstrated that, in LNCaP cells, the expression and the secretion of PAcP regulated by androgen are apparently hormone-responsive processes. Further, DHT stimulation of PAcP secretion operates within at least two levels: increased PAcP mRNA and stimulation of the secretory pathway.

Expression of human prostatic acid phosphatase activity and the growth of prostate carcinoma cells.

Human prostatic acid phosphatase (PAcP) is a tissue-specific differentiation antigen and is the major phosphotyrosyl (p-tyr) protein phosphatase in normal differentiated prostate epithelial cells. In prostate carcinomas, cellular PAcP has a low expression. We examined the expression of cellular PAcP activity and its correlation with cell growth that may lead us to understand the role of tyrosine phosphorylation in human prostate cells. LNCaP cells, which expressed the highest cellular PAcP activity, had the slowest growth rate and the lowest p-tyr level among three human prostate carcinoma cell lines: LNCaP, DU145, and PC-3. This inverse correlation was further examined in LNCaP cells, since these cells remain hormone-sensitive. Androgen, a classical stimulator of prostate cells, stimulated the growth of LNCaP cells while cellular PAcP activity decreased and p-tyr levels increased. This phenomenon was also observed when cells were treated with epidermal growth factor and fetal bovine serum. Both epidermal growth factor and fetal bovine serum stimulated the growth of LNCaP cells whereas cellular PAcP activity decreased. Furthermore, when cell growth was arrested at low temperatures (23 degrees C), cellular PAcP activity was elevated. To establish the relationship of cellular PAcP activity with cell growth rate, we transfected a complementary DNA encoding the full length PAcP protein into another human prostate carcinoma line, PC-3, that lacks endogenous PAcP. Two stable transfectants, designated PC-18 and PC-416 cells, were obtained and shown to express PAcP mRNA transcribed from the transfected complementary DNA. The expression of PAcP activity in PC-416 cells, but not PC-18 cells, was associated with a lower p-tyr level and a slower growth rate than control cells transfected with the expression vector alone. In conclusion, in LNCaP cells, the stimulated cell growth is associated with an increased p-tyr level and a decreased cellular PAcP activity. In PAcP complementary DNA-transfected PC-416 cells, the low level of p-tyr corresponds to a slow growth rate.

Acquisition of insulin-dependent protein tyrosine kinase activity during Drosophila embryogenesis.

We have previously reported (Petruzzelli, L., Herrera, R., Garcia, R., and Rosen, O. M. (1985) Cancer Cells 3, 115-121) that adult Drosophila melanogaster contain a specific, high-affinity insulin-binding protein. Insulin-dependent protein tyrosine kinase activity has now been identified in Drosophila. Activity first appears at 6-12 h of embryogenesis, increases during the 12-18-h period and falls to low levels in the adult. 125I-insulin was cross-linked specifically and with high affinity to a protein (Mr = 135,000) throughout embryogenesis and in the adult. However, during the 6-12- and 12-18-h periods of embryogenesis when insulin-dependent protein tyrosine kinase activity is expressed, another protein (Mr = 100,000) becomes cross-linked to 125I-insulin. Crosslinking to both proteins was competitively inhibited by the addition of 100 nM insulin. We conclude that the insulin-binding and insulin-dependent protein tyrosine kinase activities of the mammalian insulin receptor are conserved in Drosophila. However, the insulin-dependent protein tyrosine kinase activity of the receptor is detected only during specific times in embryogenesis.

Preparation and biological activity of taxol acetates.

Synthesis and biological activity of 2'-acetyltaxol and 7-acetyltaxol are reported. Activity is measured in vivo by cytotoxicity toward the macrophage-like cell line J774.2, and in vitro by promotion of microtubule assembly in the absence of exogenous GTP. Addition of an acetyl moiety at C-2' results in loss of in vitro activity but not cytotoxicity. The properties of 7-acetyltaxol are similar to those of taxol in its effects on cell replication and on in vitro microtubule polymerization. Therefore a free hydroxyl group at C-7 is not required for in vitro activity and this position is available for structural modifications.