Tumor-suppressive effects of neutral endopeptidase in androgen-independent prostate cancer cells.

Expression of neutral endopeptidase (NEP) 24.11 is diminished in metastatic, androgen-independent prostate cancers (PCs; C. N. Papandreou et al., NAT: MED:, 4: 50--57, 1998). To determine the effects on androgen-independent PC cells of overexpressing cell-surface NEP, an inducible tetracycline-regulatory gene expression system was used to stably introduce and express the NEP gene in androgen-independent TSU-Pr1 cells generating WT-5 cells, which expressed high levels of enzymatically active NEP protein when cultured in the absence of tetracycline. TN12 cells, which contain the identical vectors without the NEP gene and do not express NEP, were used as control. Expression of NEP in WT-5 cells after removal of tetracycline from the media resulted in a >80% inhibition in cell proliferation over a 1-week period (P < 0.005) compared with control cells. Tumor formation occurred in the prostate glands of orthotopically injected athymic mice killed at 30 days in 4 of 5 mice that were given injections of 2 x 10(6) WT-5 cells and were fed doxycycline (NEP suppressed), and in all mice that were given injections of TN12 cells and were fed with or without doxycycline. In contrast, only 1 of 5 mouse prostates developed a tumor in mice that were given injections of WT-5 cells and that did not receive doxycycline. Analysis of the mechanisms of NEP-induced growth suppression revealed that NEP expression in WT-5 cells induced a 4-fold increase in the number of PC cells undergoing apoptosis, and increased the expression of p21 tumor suppressor gene protein and the level of unphosphorylated retinoblastoma protein as determined by Western blot. Flow cytometric analysis show that induced NEP expression in WT-5 cells resulted in a G(1) cell cycle arrest. These data show that NEP can inhibit PC cell growth and tumorigenicity and suggest that NEP has potential as therapy for androgen-independent PC.

Bryostatin 1 induces prolonged activation of extracellular regulated protein kinases in and apoptosis of LNCaP human prostate cancer cells overexpressing protein kinase calpha.

Previously, we reported that 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced apoptosis of LNCaP human prostate cancer cells was accompanied by prolonged translocation of protein kinase C (PKC)alpha to non-nuclear membranes and that TPA-resistant LNCaP cells had down-regulated PKCalpha. Here we show that 10 nM bryostatin 1 induced transient membrane translocation and down-regulation of PKCalpha, prolonged translocation of PKCdelta and epsilon to non-nuclear membranes, and did not induce cell death but blocked TPA-induced apoptosis. To test the hypothesis that inhibition of TPA-induced apoptosis by bryostatin 1 was due to down-regulation of PKCalpha, we inducibly overexpressed PKCalpha in LNCaP cells. Overexpression of PKCalpha alone did not induce apoptosis, even in clones that contained much more membrane-bound, active PKCalpha than was observed in TPA-treated untransfected LNCaP cells. However, the addition of 10 nM bryostatin 1 to PKCalpha-overexpressing LNCaP cells did not yield down-regulation of PKCalpha and induced extensive apoptosis. Immunoblot analysis revealed that TPA induced prolonged hyperphosphorylation of Raf-1 and activation of extracellular-regulated/mitogen-activated protein kinases 1 and 2 in untransfected LNCaP cells, as did bryostatin 1 in PKCalpha-overexpressing cells. On the other hand, bryostatin 1 induced only transient hyperphosphorylation of Raf-1 and activation of extracellular-regulated/mitogen-activated protein kinases 1 and 2 in untransfected LNCaP cells. These results confirm a role of prolonged membrane-associated PKCalpha in PKC activator-mediated LNCaP apoptosis and suggest involvement of the mitogen-activated protein kinase pathway.

Rat protein kinase c zeta gene contains alternative promoters for generation of dual transcripts with 5'-end heterogeneity.

Protein kinase C (PKC) zeta is a phospholipid-dependent serine/threonine kinase that appears to perform important cell signaling functions. Two forms of PKC zeta RNA, with different 5' ends, have been reported. The major form (zeta) is expressed in most, if not all tissues, while the minor form (zeta'), which encodes the catalytic domain of the enzyme without most of its regulatory domain, is predominant in normal brain and certain rat prostate tumors. We report here the structure of the 5' end of the rat PKC zeta gene, demonstrating that both forms of RNA can be transcribed from the same gene through the use of alternative promoters and splicing. In luciferase reporter constructs, progressive deletions of the PKC zeta and zeta' 5' flanking sequences yielded activities that were higher in the cell lines expressing endogenous PKC zeta and zeta' RNAs, respectively. Also, multiple PCRs across different introns of the PKC zeta gene indicate that recent duplication of the gene or the existence of a closely related pseudogene in the rat genome are unlikely.

E-cadherin mediates aggregation-dependent survival of prostate and mammary epithelial cells through the retinoblastoma cell cycle control pathway.

E-cadherin and the retinoblastoma tumor suppressor (Rb) are traditionally associated with diverse regulatory aspects of cell growth and differentiation. However, we have discovered new evidence, which suggests that these proteins are functionally linked in a physiologic pathway required for cell survival and programmed cell death. Pharmacological activation of protein kinase C (PKC) or inducible overexpression and activation of the alpha isozyme of PKC (PKCalpha) resulted in approximately 60% apoptosis of mammary and prostate epithelial cells. Interestingly, the surviving cells had undergone dramatic aggregation concurrent with increased E-cadherin expression. When aggregation was inhibited by the addition of an E-cadherin-blocking antibody, apoptosis increased synergistically. We hypothesized that survival of the aggregated population was associated with contact-inhibited growth and that apoptosis might result from aberrant growth regulatory signals in non-aggregated, cycling cells. This hypothesis was confirmed by experiments that demonstrated that E-cadherin-dependent aggregation resulted in Rb-mediated G1 arrest and survival. Immunoblot analysis and flow cytometry revealed that hypophosphorylated Rb was present in non-aggregated, S phase cultures concurrent with synergistic cell death. We have also determined that the loss of membrane E-cadherin and subsequent hypophosphorylation of Rb in luminal epithelial cells preceded apoptosis induced by castration. These findings provide compelling evidence that suggests that E-cadherin-mediated aggregation results in Rb activation and G1 arrest that is critical for survival of prostate and mammary epithelial cells. These data also indicate that Rb can initiate a fatal growth signal conflict in non-aggregated, cycling cells when the protein is hypophosphorylated as these epithelial cells enter S phase.

Mapping, genomic organization and promoter analysis of the human prostate-specific membrane antigen gene.

Prostate-specific membrane antigen (PSMA) is a 100 kDa type II transmembrane protein with folate hydrolase and NAALAdase activity. PSMA is highly expressed in prostate cancer and the vasculature of most solid tumors, and is currently the target of a number of diagnostic and therapeutic strategies. PSMA is also expressed in the brain, and is involved in conversion of the major neurotransmitter NAAG (N-acetyl-aspartyl glutamate) to NAA and free glutamate, the levels of which are disrupted in several neurological disorders including multiple sclerosis, amyotrophic lateral sclerosis, Alzheimer's disease and schizophrenia. To facilitate analysis of the role of PSMA in carcinoma we have determined the structural organization of the gene. The gene consists of 19 exons spanning approximately 60 kb of genomic DNA. A 1244 nt portion of the 5' region of the PSMA gene was able to drive the firefly luciferase reporter gene in prostate but not breast-derived cell lines. We have mapped the gene encoding PSMA to 11p11-p12, however a gene homologous, but not identical, to PSMA exists on chromosome 11q14. Analysis of sequence differences between non-coding regions of the two genes suggests duplication and divergence occurred 22 million years ago.

Computerized prompts for cancer screening in a community health center.

BACKGROUND: We describe the implementation and subsequent use of a computerized health maintenance tracking system in a large, urban, North Carolina community health center (Lincoln Community Health Center) as part of a larger study designed to increase rates of mammography, Papanicolaou tests, and smoking cessation in low-income African-Americans. METHODS: Clinicians from the Lincoln Community Health Center were involved in the design and implementation of the computer system. At each office visit, clinicians received a computerized encounter form indicating needed screening tests, counseling, and immunizations for each randomly selected study patient (n = 1318). RESULTS: Initial clinician compliance rates with filling out the form were 95 percent (mammography), 82 percent (Papanicolaou test), 77 percent (clinician breast examination), and 55 percent (smoking cessation). Cumulative compliance leveled off at 21 months to 65 percent, 57 percent, 53 percent, and 38 percent, respectively, despite multiple reminder strategies. When surveyed, most clinicians thought it was a good reminder system but said they did not always complete the form because of time demands. Costs of adapting and implementing the system were $23,332.08 ($17.70 per study). Per-patient costs would have been reduced further if more patients had been included in the project. CONCLUSIONS: State-of-the-art computer prompting systems can be useful in a community health center; however, even with prompting, clinicians still only addressed health maintenance with their patients about 50 percent of the time. Additional interventions will be needed, particularly in low-income populations, to meet the Healthy People 2000 goals in health promotion.

Neutral endopeptidase 24.11 loss in metastatic human prostate cancer contributes to androgen-independent progression.

Neutral endopeptidase 24.11 (NEP) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). We report that NEP expression and catalytic activity are lost in vitro in androgen-independent but not androgen-dependent PC cell lines. In vivo, NEP protein expression is commonly decreased in cancer cells of metastatic PC specimens from patients with androgen-independent but not androgen-dependent PC. Overexpression of NEP in androgen-independent PC cells or incubation with recombinant NEP inhibits PC cell growth. Furthermore, in androgen-dependent PC cells, expression of NEP is transcriptionally regulated by androgen and decreases with androgen withdrawal. These data suggest that decreased NEP expression, common in androgen-independent PCs, is facilitated by the elimination of androgens, and that NEP loss plays an important role in the development of androgen-independent PC by allowing PC cells to use mitogenic neuropeptides as an alternate source to androgen in order to stimulate cell proliferation.

Evaluation of the tetracycline-repressible transactivator system for inducible gene expression in human prostate cancer cell lines.

BACKGROUND: Studies of genes that may inhibit growth or induce death of cells are facilitated greatly by tightly controlled expression of those genes. A promising system for control of transgene expression over a wide range is the tetracycline-repressible transactivator (tTA) system developed by Gossen and Bujard [Proc Natl Acad Sci USA 1992;89:5547-5551]. We investigated the effectiveness of this system in three well-established human prostate cancer cell lines. METHODS: LNCaP, PC-3, and Tsu-Pr1 cells were transfected with a vector coding for the tTA protein and/or a luciferase reporter vector, and luciferase activity was measured in the presence and absence of tetracycline or the tTA protein. RESULTS: In the absence of tetracycline, the tTA system yielded high levels of luciferase activity in all three cell lines. Background luciferase activity in the presence of tetracycline was nearly undetectable in LNCaP cells, moderate in Tsu-Pr1 cells, and more than 20-fold higher in PC-3 than in Tsu-Pr1 cells. Similar background activity was observed in Tsu-Pr1 and PC-3 cells, even in the absence of the transactivator protein. CONCLUSIONS: The tTA system should be useful for stable transfection of cytotoxic transgenes in LNCaP cells and for control of transgene expression over a wide range in Tsu-Pr1 and PC-3 cells.

Retinoblastoma protein-dependent growth signal conflict and caspase activity are required for protein kinase C-signaled apoptosis of prostate epithelial cells.

Both protein kinase C and the retinoblastoma tumor suppressor protein have been linked to the regulation of cell growth and cell death, suggesting the differential roles these factors play in mediating cell fate. In some cells, protein kinase C-induced activation of the retinoblastoma protein results in G1 arrest. However, inducible overexpression and activation of the protein kinase Calpha isozyme or the addition of 12-O-tetradecanoylphorbol-13-acetate in the prostate epithelial cell line, LNCaP, resulted in apoptosis preceded by induction of p21 and dephosphorylation of the retinoblastoma protein. Consistent with a role for the retinoblastoma growth suppressor protein in protein kinase C-induced apoptosis, DU145 cells, which do not express functional retinoblastoma protein or LNCaP cells, which have been transfected with the retinoblastoma inhibitor, E1a, were resistant to apoptosis. LNCaP apoptosis was initiated by a unique conflict between the growth-suppressive activity of the retinoblastoma protein and growth-promoting mitogenic signals. Thus, when this conflict was prevented by serum depletion, apoptosis was suppressed. The caspase family of cysteine proteases is believed to encompass the execution machinery of mammalian apoptosis, and addition of the cell-permeable caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, afforded nearly total protection from protein kinase C-signaled apoptosis. This protection correlated with the total loss of caspase activity as measured by the proteolytic cleavage of nuclear poly(ADP-ribose) polymerase. On the basis of these results, we propose that protein kinase C regulates a novel cell death pathway that is initiated by a cellular conflict between retinoblastoma growth-suppressive signals and serum mitogenic signals in proliferating prostate epithelial cells and that is executed by the caspase family of cysteine proteases.

Readiness to change smoking behavior in a community health center population.

This study examines predictors of readiness to change smoking behavior in a sample of smokers who receive care at a community health center that serves a predominantly low income African American population. Prior to initiating interventions we conducted a telephone survey with a random sample of 1318 adult users who had visited the center in the last 18 months; 379 (28.8%) were current smokers (40.3% of males, 23.9% of females, 42.7% of Whites, and 25.3%, of African Americans). Multiple logistic regression analysis showed nine factors significantly associated with readiness to change smoking behavior: male gender; a previous quit attempt; a perception of risk of lung cancer from smoking; greater desire to quit smoking; a perception that smoking bothers others; doctor advice to stop smoking at last health visit; records kept for scheduling doctor appointments; thinking that losing a pleasure would not be a problem if quit smoking; and poorer self-reported health status. These findings provide direction for developing interventions for similar low income, high risk populations. The results indicate that it may be useful to heighten awareness of the risks of smoking and to assure that smokers receive clear quit smoking messages from their providers. Women need special attention since they are less ready to quit than men.

Colorectal screening patterns and perceptions of risk among African-American users of a community health center.

This study investigated risk perceptions and screening patterns for colorectal cancer among predominately low-income African-Americans age 50 and older who used a community health center. The majority of respondents either rated their risk as below average (36%) or did not know their risk (37%) for colorectal cancer. Individuals who provided a risk estimate versus those who did not know their risk were younger and held more accurate beliefs about colorectal cancer. Attributions of perceived risk best distinguished respondents who evaluated their risk to be below average versus average and above average. Compared to respondents who could not explain their risk, those who provided psychological, personal action, and heredity causes were more likely to view their risk as below average than average or above average. In comparison to national norms, these subjects reported higher frequencies of ever having had a digital rectal exam (DRE, 90%), fecal occult blood test (FOBT, 75%) and sigmoidoscopy (SIG, 33%). Moreover, 63%, 53%, and 81% reported their most recent screening for DRE, FOBT, and SIG, respectively, in accordance with ACS and NCI recommended guidelines. However, a subsequent medical audit failed to confirm these self-reports. These results suggest that: 1) educational efforts are needed to enhance knowledge and accuracy of risk perceptions for colorectal cancer; 2) further studies on attributions of risk are needed that may prove useful for developing intervention programs, and 3) studies need to interpret self-report data for colorectal cancer with caution.

Cancer screening practices among women in a community health center population.

BACKGROUND: Cancer takes a disproportionate toll on disadvantaged Americans. Poverty and low education are risk factors for underuse of cancer screening. METHODS: In this report, we discuss predictors of adherence to cancer screening (mammography, clinical breast exam [CBE], and Pap tests) among 926 women who receive care at a community health center that serves a predominantly low-income and minority population. We examine predictors for each of the tests and for a composite measure of overall cancer screening test compliance. In studying multiple screening behaviors we not only investigate factors associated with each individual behavior, but we also identify consistently effective factors across several behaviors. RESULTS: The analysis indicates consistent effects of age, education, and insurance status on cancer screening. In addition, decisional balance, a measure of a person's beliefs about the pros and cons of complying with the screening test, is associated strongly with adherence. We have extended earlier findings about the positive relationship between decisional balance and mammography to include decisional balance and Pap tests, as well. This finding suggests that behavioral interventions that target decisional balance can effectively promote adherence to cancer screening tests.

Overexpression of protein kinase C-zeta (PKC-zeta) inhibits invasive and metastatic abilities of Dunning R-3327 MAT-LyLu rat prostate cancer cells.

Previously, we reported that protein kinase C (PKC)-zeta mRNA levels are reduced markedly in metastatic Dunning R-3327 rat prostate tumors relative to the nonmetastatic Dunning H tumor and normal rat prostate (C.T. Powell et al., Cell Growth & Differ., 5: 143-149, 1994). To examine the effect of PKC-zeta on metastatic and invasive abilities of an aggressive Dunning R-3327 cell line, we generated stably transfected clones of MAT-LyLu cells that overexpress active PKC-zeta. PKC-zeta-overexpressing MAT-LyLu cells exhibited tumorigenicity and growth rates in syngeneic rats similar to those of MAT-LyLu cells transfected with vector alone or untransfected MAT-LyLu. However, nine independent clones of PKC-zeta-expressing cells exhibited an average 2-fold lower tendency to metastasize to lungs relative to vector-transfected MAT-LyLu cell clones, with about 2-fold and 4.5-fold fewer metastases per rat in two separate protocols. In addition, the ability of four PKC-zeta overexpressing MAT-LyLu clones to invade through Matrigel in a Boyden chamber assay was reduced an average of 12-fold relative to three vector-transfected clones. These results indicate that increased PKC-zeta expression can substantially suppress invasion and metastasis by an aggressive rat prostate tumor.

Persistent membrane translocation of protein kinase C alpha during 12-0-tetradecanoylphorbol-13-acetate-induced apoptosis of LNCaP human prostate cancer cells.

Others have reported that the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA), an activator and down-regulator of most protein kinase C (PKC) isozymes, can induce apoptotic cell death of androgen-sensitive LNCaP but not androgen-insensitive PC-3 or DU 145 human prostate cancer cells. As a first step toward uncovering the mechanism by which TPA induces apoptosis of LNCaP cells, we quantified expression of PKC isozyme mRNAs in unmodified and TPA-resistant LNCaP cells and in naturally TPA-resistant PC-3, PC-3M, and DU 145 cells. All of the cell lines and normal prostate expressed RNAs for PKC alpha, delta, epsilon, eta, and mu; only DU 145 cells and normal prostate expressed PKC beta and theta RNAs, and none expressed PKC gamma. The amount of PKC alpha RNA and protein was 6- to 38-fold lower, and PKC mu RNA was 4.5- to 16.5-fold higher in unmodified and TPA-resistant LNCaP cells than in the androgen-independent cells. We examined the effects of TPA on PKC alpha and mu mRNA levels and on membrane translocation of PKC alpha. Incubation with TPA for 6 h or more induced 95% inhibition of cell growth, a transient 12-fold increase and 5-fold decrease in PKC alpha and mu mRNA levels, respectively, and prolonged translocation of PKC alpha to non-nuclear membranes in unmodified LNCaP cells and in TPA-resistant LNCaP cells from which TPA had been removed for 10 days. TPA-resistant LNCaP cells in the continuous presence of TPA, or 24 h after removal of TPA, had down-regulated PKC alpha and remained resistant to re-addition of TPA. These data demonstrate a strong correlation of the presence and absence of membrane PKC alpha with apoptosis and resistance to apoptosis, respectively.

Alternatively spliced variants of prostate-specific membrane antigen RNA: ratio of expression as a potential measurement of progression.

We examined expression of prostate-specific membrane antigen (PSM) mRNA in normal prostate using reverse transcription-PCR and sequencing. An alternatively spliced variant, PSM', along with the previously described PSM form, was found in normal prostate. PSM' cDNA is shorter (2387 nucleotides) than PSM (2653 nucleotides). The cDNAs are identical except for a 266-nucleotide region near the 5' end of PSM cDNA (nucleotide 114-380) that is absent from PSM'. This deleted region includes the translation initiation codon and codons for the putative transmembrane domain of PSM. Thus, PSM' RNA codes for a protein that has no apparent signal sequence. We verified the existence of spliced mRNA variants in human primary tissue specimens by RNase protection assay. In LNCaP human prostatic cancer cells and in primary prostate tumors, PSM is the dominant form. In contrast, normal human prostate expressed more PSM' than PSM. Benign prostatic hypertrophy samples showed about equal expression of both variants. We quantified the relative expression of each variant by densitometry and compiled a tumor index, which is the ratio of PSM:PSM' level. LNCaP has an index ranging from 9-11, carcinoma of the prostate from 3-6, benign prostatic hypertrophy from 0.75-1.6, and normal prostate from 0.075-0.45. The index reflects the increased expression of PSM over PSM' following the progression from normal to tumor state. This tumor index may be a useful indicator for the measurement of tumor progression. PSM and PSM' may be functionally different proteins as a result of differences in structure or cellular location. We are investigating the prevalence of one form over the other and how it may influence tumor progression.

Sensitive detection of prostatic hematogenous tumor cell dissemination using prostate specific antigen and prostate specific membrane-derived primers in the polymerase chain reaction.

We developed a polymerase chain reaction based assay enabling sensitive detection of hematogenous tumor cell dissemination in patients with prostate cancer. We performed "nested polymerase chain reaction," amplifying messenger ribonucleic acid sequences unique to prostate specific antigen (PSA) and to the prostate specific membrane antigen, and compared the respective results. Prostatic tumor cells were detected in 2 of 30 patients (6.7%) by polymerase chain reaction with PSA derived primers, while prostate specific membrane primers detected tumor cells in 19 (63.3%). All 16 negative controls had negative PSA and prostate specific membrane polymerase chain reaction. Assays were repeated to confirm results, and polymerase chain reaction products were verified by deoxyribonucleic acid sequencing and Southern analysis. Patients harboring circulating prostatic tumor cells as detected by prostate specific membrane and not by PSA polymerase chain reaction included 7 of 13 previously treated by radical prostatectomy who had nonmeasurable serum PSA levels at the time of this assay. The significance of these findings with respect to future disease recurrence and progression will be investigated.

Differential expression of protein kinase C isoforms in human colorectal cancers.

Protein kinase C (PKC), a serine/threonine kinase central to signal transduction, is implicated in tumor promotion. At present, 10 PKC isoforms have been cloned but their precise tissue-specific role has yet to be defined. In order to determine if PKC is reduced in colorectal cancers (CRC) and if specific PKC isoforms are altered in different stages of human CRC progression, total RNA was extracted from human primary CRC, liver metastases, paired normal mucosa, and liver as well as CRC cell lines and examined for specific PKC isoform mRNA expression. PKC-alpha, beta II, delta, epsilon, eta(L), theta, and zeta were expressed in all tissues examined, while PKC-beta I was not detected. PKC-alpha, beta II, delta, epsilon, and zeta were decreased in most primary CRC. However, the reduction in PKC-beta II was greatest in advanced primary CRC (P < 0.05). Although PKC-gamma was detected in about 29.6% of primary CRC and liver metastases, it was absent from all corresponding normal tissue. In addition, a second band hybridizing to our PKC-gamma probe was uniquely present only in cancerous tissue and not in brain control, suggestive of alternative splicing. PKC-alpha, delta, epsilon, and zeta were present in all cell lines. PKC-beta I/II were both uniformly absent from all cell lines. Since mRNA expression for most PKC isoforms is decreased in CRC, the previously reported decreases in overall PKC activity in CRC are not solely due to a post-translational enzyme modification.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal by transferrin of growth-inhibitory effect of suramin on hormone-refractory human prostate cancer cells.

BACKGROUND: The second leading cause of cancer-related deaths in American men is metastatic hormone-refractory adenocarcinoma of the prostate, for which there is currently no effective treatment. Transferrin is abundant in bone stroma and has been found to stimulate models of hormone-refractory metastatic prostate cancer. Suramin, a compound that has been used to treat metastatic prostate cancer, has been demonstrated to antagonize the binding of transferrin to the transferrin receptor and to suppress uptake of iron by hematopoietic cells. PURPOSE: The purpose of our study was to determine whether transferrin may reverse the inhibitory action of suramin on metastatic prostate-derived cell lines. METHODS: Five human prostate cell lines (PC-3, PC-3M, DU-145, TSU-Pr1, and LNCaP) derived from metastatic deposits were examined for response to growth stimulation by apotransferrin, for the presence of transferrin receptors by binding of 125I-labeled transferrin, and for relative transferrin receptor messenger RNA (mRNA) content by ribonuclease protection assays. We measured the amount of growth inhibition by suramin in low serum assays to demonstrate maximal inhibition over the apotransferrin to reverse the inhibition of suramin in these tumors. RESULTS: The results clearly demonstrate that the androgen-insensitive metastatic cell lines (PC-3, PC-3M, DU-145, and TSU-Pr1) demonstrate increased cell numbers when exposed to holotransferrin or apotransferrin, while the androgen-sensitive cell line (LNCaP) did not show any increase. All cell lines demonstrated a similar number of transferrin receptors and transferrin receptor mRNA. We used these maximally inhibitory, but clinically relevant, concentrations of suramin to determine whether transferrin could reverse the inhibition, and it did, but only in the androgen-insensitive metastatic lines. Indeed, in the PC-3 cells, inhibition turned to stimulation with the addition of transferrin, and even at the highest concentration of suramin tested, 400 microM, a concentration that would be toxic to patients, the amount of inhibition by suramin was still reduced by more than 50% by transferrin in TSU-Pr1 cells. In the androgen-sensitive LNCaP cells, however, transferrin had limited ability to block the inhibitory activity of suramin. CONCLUSIONS: Concentrations of tumor-stimulating factors, such as transferrin, in the metastatic microenvironment need to be taken into consideration in the use of suramin and suramin-like derivatives. Novel strategies need to be identified that will negate the action of transferrin on androgen-insensitive cells.

Sensitive nested reverse transcription polymerase chain reaction detection of circulating prostatic tumor cells: comparison of prostate-specific membrane antigen and prostate-specific antigen-based assays.

A highly sensitive nested reverse transcriptase-PCR assay, with primers derived from the prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) cDNA sequences, has been used to detect occult hematogenous micrometastatic prostate cells. In 77 patients with prostate cancer, PSM and PSA primers detected circulating prostate cells in 48 (62.3%) and 7 (9.1%) patients, respectively. In treated stage D disease patients, PSM primers detected cells in 16 of 24 patients (66.7%), while PSA primers detected cells in 6 of 24 (25%). In post-radical prostectomy patients with negative serum PSA values, PSM primers detected metastases in 21 of 31 patients (67.7%), whereas PSA primers detected cells in only 1 of 33 (3.0%), indicating that micrometastatic spread may be a relatively early event in prostate cancer. The analysis of 40 individuals without known prostate cancer provides evidence that this assay is highly specific and suggests that PSM expression may predict the development of cancer in patients without clinically apparent prostate cancer. Using PSM primers, we detected micrometastases in 4 of 40 controls, 2 of whom had known benign prostatic hyperplasia and were later found to have previously undetected prostate cancer. The clinical significance of detection of hematogenous micrometastic prostate cells using PSM primers and potential applications of this molecular assay, as well as the assay for PSA, merit further study.

Overexpression of protein kinase C beta I in a murine keratinocyte cell line produces effects on cellular growth, morphology and differentiation.

The present study demonstrates that the murine keratinocyte cell line 3PC expresses the Ca(2+)-insensitive isoforms of protein kinase C (PKC) delta, epsilon, zeta and (at both the mRNA and protein levels), but does not express the Ca(2+)-sensitive PKC isoforms alpha, beta or gamma. Recombinant retroviral gene transduction was used to develop derivatives of this cell line that stably express high levels of 1 PKC beta I-related transcripts and proteins, and have increased levels of Ca(2+)-stimulated PKC enzyme activity. Functional overexpression of the PKC beta I isoform in 3PC cells enhances both 12-O-tetradecanoyl phorbol-13-acetate-induced growth inhibition, and Ca(2+)-induced morphologic differentiation.