Anticancer Drug-mediated Induction of Multidrug Resistance-associated Genes and Protein Kinase C Isozymes in the T-Lymphoblastoid Cell Line CCRF-CEM and in Blasts from Patients with Acute Lymphoblastic Leukemias

The major determinants mediating drug resistance in acute lymphoblastic leukemias (ALL) unresponsive to chemotherapy, are still unclear. For example, it is still unknown whether selection or induction processes are responsible for drug resistance here or whether protein kinase C (PKC) isozymes contribute to the resistant phenotype. Therefore, inducibility of resistance factors or PKC isozymes genes was examined in CCRF‐CEM cells treated with diverse anticancer drugs‐ adriamycin, camptothecin, etoposide or vincristine‐at sublethal concentrations for 24 h. MDR1, MRP1, LRP and PKC isozyme α, β₁, β₂, ε, ι, η, θ, ζ gene expression was determined by cDNA‐PCR. We found significant dose‐dependent, mostly combined, induction of the MDR1, MRP1 and LRP genes. Significantly enhanced gene expression of the majority of PKC isozyme genes was found after treatment with camptothecin. PKCζ was upregulated throughout by each anticancer drug applied in this setting. A series of selected CCRF‐CEM‐derived multidrug resistance (MDR) sublines also showed enhanced expression of the PKC isozymes compared to the parental cell line. MDR1 and PKCη gene expression levels were correlated highly significantly. Blasts from two patients with ALL during the first week of monotherapy with steroids revealed combined induction of the MDR1, multidrug resistance‐associated protein 1 (MRP1), lung cancer resistance‐related protein (LRP) and most PKC isozymes, predominantly PKCζ. Another patient with T‐ALL, who failed to respond to four months of intensive chemotherapy, showed an enhanced MRP1 gene expression combined with markedly overexpression of PKCη and PKCθ. Furthermore, the camptothecin and etoposide‐mediated induction of resistance factors in the CCRF‐CEM cell line could be suppressed by staurosporine, a rather unspecific inhibitor of protein kinases. However, selective inhibitors of PKC isozymes (bisindolylmaleimide GÖ 6850, indolocarbazole GÖ 6976) produced no significant effects here. Therefore, the PKC isozymes η, θ and ζ are of interest as potential targets to overcome drug resistance in ALL.     

Effects of increased expression of protein kinase C on radiation-induced cell transformation

The in vitro oncogenic transformation of C3H 10T1/2 cells byionizing radiation is known to be enhanced by the tumor promoter12-O-tetradecanoylphorbol-13-acetate (TPA). It is also knownthat the activation of protein kinase C (PKC) by TPA is an importantstep in its tumor-promoting effect. In the present study, weexamined the effects of overexpression of a specific isoformof PKC, PKC_ß1 on 7-ray-induced transformation of 10T1/2cells. In addition, the effects of overexpression of PKC_ß1on the malignant phenotype of a previously transformed 10T1/2cell line were also evaluated. Derivatives of 10T1/2 cells thatstably overexpress PKC_ß1 were obtained by transductionwith the retroviral expression vector pMV7 carrying the ratPKC_ß1 cDNA sequence. We found that the parental 10T1/2cells and a control cell line 10T1/2 MV7, which carried onlythe pMV7 vector without the cDNA insert, expressed dose-dependenttransformation frequencies when exposed to 7-rays. On the otherhand, concurrently treated PKC-overexpressing cells that hadan 11-fold increase in enzyme activity (PKC-4 cells) failedto yield any morphologically identifiable foci. Cell lines thatexpressed lower levels of PKC_ß1 were partially resistantto transformation by     

Association of protein kinase C activation with induction of ornithine decarboxylase in murine but not human keratinocyte cultures

The goal of this study was to compare the response of mouse epidermal keratinocytes (MEKs) and human epidermal keratinocytes (HEKs) to 12-O-tetradecanoylphorbol-13-acetate (TPA) with respect to the activation and downregulation of protein kinase C (PKC), the expression of c-jun and c-fos, and the expression and induction of ornithine decarboxylase (ODC) activity. Keratinocytes from adult CD-1 mice and from discarded adult human skin were grown in primary culture in a high-calcium serum-free medium that supported proliferation and differentiation. Immunoblotting of freshly isolated and cultured MEKs and HEKs for isozymes of protein kinase C revealed that fresh HEKs contained PKCα, PKCβ, and PKCδ; no PKCγ, PKC?, or PKCζ were detected. In fresh MEKs, PKCα, PKCβ, PKCΔ, and PKCζ were observed, but not PKCγ or PKCζ. After 2 wk in culture, the isozyme profiles of MEKs and HEKs were similar except that PKCγ was noticeably present in HEK cultures. Activation of partially purified total PKC by TPA was similar in freshly isolated and cultured MEKs and HEKs, indicating that the two species were similar in this regard and that 2 wk of culture did not alter this characteristic. When MEK and HEK cultures were treated with TPA for 3 h, less than 30% of the control level of PKC activity was detected, indicating that TPA-induced downregulation of PKC was similar in MEKs and HEKs. After treatment with TPA, MEK cultures produced a large induction of both c-jun and c-fos mRNA by 60 min, as determined by northern blot analysis, and a large induction of ODC mRNA and enzyme activity by 6 h. TPA treatment of cultured HEKs, however, did not induce ODC activity; in fact, less activity, compared with that of control cultures, was observed. Northern blot analysis also revealed no increase in c-jun, c-fos, and ODC mRNA in HEKs. However, c-jun and c-fos mRNA and both ODC mRNA and enzyme activity were induced in HEKs fed growth factors after several days of deprivation. This suggests that the lack of ODC induction by TPA in HEKs is probably due to species differences in downstream steps in PKC signal transduction.     

Tamoxifen induces selective membrane association of protein kinase C epsilon in MCF-7 human breast cancer cells

Tamoxifen, a synthetic antiestrogen, is known for its antitumoral action in vivo; however, it is well accepted that many tamoxifen effects are elicited via estrogen receptor-independent routes. Previously, we reported that tamoxifen induces PKC translocation in fibroblasts. In the present study, we investigated the influence of tamoxifen, and several triphenylethylene derivatives, on protein kinase C (PKC) in MCF-7 human breast cancer cells. As measured by Western blot analysis, tamoxifen elicited isozyme-specific membrane association of PKC-ϵ, which was time-dependent (as early as 5 min post-treatment) and dose-dependent (5.0–20 μM). Tamoxifen did not influence translocation of α, β, γ, δ or ζ PKC isoforms. Structure-activity relationship studies demonstrated chemical requirements for PKC-ϵ translocation, with tamoxifen, 3-OH-tamoxifen and clomiphene being active. Compounds without the basic amino side chain, such as triphenylethylene, or minus a phenyl group, such as N,N-dimethyl-2-[(4-phenylmethyl)phenoxy]ethanamine, were not active. In vitro cell growth assays showed a correlation between agent-induced PKC-ϵ translocation and inhibition of cell growth. Exposure of cells to clomiphene resulted in apoptosis. Since PKC-ϵ has been associated with cell differentiation and cellular growth-related processes, the antiproliferative influence of tamoxifen on MCF-7 cells may be related to the interaction with PKC-ϵ. Int. J. Cancer 77:928–932, 1998.© 1998 Wiley-Liss, Inc.     

Do Structural Changes of T Cell Receptor Complex Occur in Tumor-bearing State?

T cells in tumor-bearing mice and cancer patients were recently shown to be devoid of CD3-ζ chain, a signal-transducing invariant chain in T cell receptor (TCR) complex, and p56^lck tyrosine kinase. In the present study, we investigated the structure and function of TCR complex in T cells from BALB/c mice bearing CSA1M fibrosarcoma. The expressions of TCR chains and p56^lck in a T cell-enriched population from spleen were analyzed. Almost complete loss of CD3-ζ and p56^lck was observed in the preparation from tumor-bearing mice as assessed by immunoblotting analysis using whole cell lysates, whereas the amounts of other TCR chains were relatively unchanged. However, these changes were due to the increase of contaminating Mac-1⁺ cells in the spleen of tumor-bearing mice because: 1) the removal of Mac-1⁺ cells led to the restoration of CD3-ζ and p56^lck; and 2) CD3-ζ was clearly present when the preparation was solubilized with ionic detergent. Fc receptor γ chain detected in the preparation from tumor-bearing mice disappeared along with the removal of Mac-1⁺ cells. These observations were further supported by the finding that addition of Mac-1⁺ cells from tumor-bearing mice to normal T cells resulted in loss of CD3-ζ, leaving CD3-ζ largely intact. When T cells from tumor-bearing mice were highly purified by depletion of Mac-1⁺ cells, these T cells contained normal amounts of CD3-ζ at mRNA, protein, and surface levels, and expressed the properly assembled TCR complex on their cell surface. Moreover, stimulation of the TCR in these T cells by anti-TCR antibodies resulted in a comparable Ca²⁺ mobilization to that observed in normal T cells. These results suggest that no structural changes occur in TCR complex in our tumor-bearing mice, and that complete depletion of Mac-1⁺ cells in important to assess the structure of TCR complex.     

Deletion analysis of protein kinase c inactivation by calphostin C

Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC-α (a Ca²⁺-dependent conventional isoform) and PKC-α (a Ca²⁺-independent novel isoform) are similarly inactivated by calphostin C (75–100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC. We therefore performed studies to identify a critical region in the regulatory domain of PKC-α required for inactivation by calphostin C. A series of N-terminal–truncation mutants of bovine PKC-α expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-α by 80–90%. This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND 140) was inactivated by only 20%. These findings imply that the aa sequence 92–140 is a structural determinant of PKC-α inactivation by calphostin C. This sequence contains one of the phorbol ester-binding sites (aa 102–144), which is highly conserved among most PKC isoforms including PKC-α. In addition to aa 92–140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca²⁺) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity. These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor. The significance of the proposed proximity effect of calphostin C action is discussed. © 1995 Wiley-Liss Inc.     

Quantification of PKC family genes in sporadic breast cancer by qRT-PCR: Evidence that PKCι/λ overexpression is an independent prognostic factor

Drugs targeting protein kinase C (PKC) show promising therapeutic activity. However, little is known about the expression patterns of the 11 PKC genes in human tumors, and the clinical significance of most PKC genes is unknown. We used qRT-PCR assays to quantify mRNA levels of the 11 PKC genes in 458 breast tumors from patients with known clinical/pathological status and long-term outcome. The proportion of tumors in which the expression of the different genes was altered varied widely, from 9.6% for PKN2 to 40.2% for PKCι/λ. In breast tumors, overexpression was the main alteration observed for PKCι/λ (33.4%), PKCδ (29.5%) and PKCζ (9.6%), whereas underexpression was the main alteration observed for PKCα (27.3%), PKCε (11.6%), PKCη (8.7%) and PKN2 (8.1%). Both overexpression and underexpression were observed for PKCβ (underexpression 15.5%, overexpression 13.8%), PKCθ (underexpression 14.8%, overexpression 10.0%) and PKN1 (underexpression 6.6%, overexpression 7.4%). Several links were found between different PKC genes; and also between the expression patterns of PKC genes and several classical pathological and clinical parameters. PKCι/λ alone was found to have prognostic significance (p = 0.043), whereas PKCα showed a trend towards an influence on relapse-free survival (p = 0.052). PKCι/λ retained its prognostic significance in Cox multivariate regression analysis (p = 0.031). These results reveal very complex expression patterns of PKC genes in breast tumors, and suggest that their expression should be considered together when evaluating anti-tumoral drugs. PKCι/λ seems to be the most promising therapeutic target in breast cancer.     

Comparison of protein kinase C activity and isoform expression in cisplatin-sensitive and -resistant ovarian carcinoma cells

Cellular sensitivity to cis-diamminedichloroplatinum(II) (cDDP) can be regulated by protein kinase C (PKC) signal transduction pathway. Activators of PKC were shown to en- hance the sensitivity of human ovarian carcinoma 2008 cells to cDDP. We have examined whether or not the PKC signal transduction pathway is affected during-development of resistance by tumor cells to cDDP. A 2-fold decrease in PKC activity was observed in cDDP-resistant ovarian carcinoma 2008 J. C13*5.25 cells compared with the drug-sensitive 2008 cells. Subceflular distribution studies revealed a reduction in both cytosolic and paniculate PKC activities in 2008/C13*5.25 cells. The pattern of PKC isoform expression was compared in cDDP-sensitive and -resistant cell lines by Western blot analysis with isoform-specific antibodies to PKC. The parental cells expressed PKCα, -ε, and -ζ isoforms. The abundance of PKCα decreased significantly in 2008/C 13*5.25 cells, whereas the amount of PKCe increased moderately in the resistant variant, with no alteration in PKCε content. Therefore, a reduction in PKCa and/or an increase in PKCε expression may be associated with the drug-resistant phenotype. © 1995 Wiley-Liss, Inc.     

mTOR complex component Rictor interacts with PKCzeta and regulates cancer cell metastasis

Epidermal growth factor (EGF) mediates breast cancer cell chemotaxis and metastasis through mechanisms that involve the growth-regulatory mammalian target of rapamycin (mTOR) complex mTORC2, but the mechanisms involved remain obscure. Here, we report that the rapamycin-insensitive mTORC2 component protein Rictor is a critical mediator of metastasis in breast cancer cells. In patients with ductal carcinoma, Rictor expression was associated with increased lymph node metastasis. EGF induced translocation and colocalization of Rictor with protein kinase Cζ (PKCζ), a pivotal molecule in chemotaxis signaling. Further, Rictor coimmunoprecipitated with PKCζ in the absence of the mTORC2 complex. Small interfering RNA-mediated knockdown of Rictor inhibited EGF-induced PKCζ phosphorylation and translocation along with phosphorylation of the key F-actin binding protein cofilin. In parallel, Rictor knockdown reduced cellular chemotactic capacity and ablated pulmonary metastasis in a xenograft mouse model of breast cancer. Our findings identify Rictor as an important mediator of chemotaxis and metastasis in breast cancer cells.     

Protein kinase C isotypes required for phorbol-ester induction of stromelysin-1 in rat fibroblasts

The phorbol-ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent inducer of the metalloproteinase stromelysin in fibroblasts in vivo and in several cultured cell lines. Rat-1 and Rat-2 fibroblasts, however, do not respond to TPA stimulation by induction of stromelysin gene activity, although collagenase promoter-mediated activity is induced threefold by TPA treatment in these cells. We determined that rat fibroblasts expressed protein kinase C (PKC) α, PKCδ, PKCϵ, and PKCζ but neither the mRNA nor the protein for PKCβ. When Rat-2 fibroblasts were stably transfected with an expression vector producing PKCβ, however, TPA treatment of these variants resulted in a 3.1-fold induction of stromelysin promoter-mediated luciferase activity compared with a 1.3-fold induction in parental Rat-2 cells (P < 0.002). Transient transfection of PKCϵ produced a small but significant increase in TPA-stimulation of both stromelysin- and collagenase-mediated gene expression. These results suggest that there are PKC isotype-specific signaling pathways that can differentially regulate matrix metalloproteinase gene expression. © 1996 Wiley-Liss, Inc.     

The role of protein kinase C isoenzymes in the growth inhibition caused by bryostatin 1 in human A549 lung and MCF-7 breast carcinoma cells

Bryostatin I is a natural product currently under clinical evaluation as an antitumor agent. Like the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13 -acetate (TPA) it activates protein kinase C (PKC). Bryostatin I inhibits the growth of the human-derived A549 lung and MCF-7 adenocarci-noma cell lines, but much more weakly than TPA. The hypotheses were tested that differences between cell lines in their response to bryostatin I are related to cellular PKC isotype content, and that differences between TPA and bryostatin I in their effects on cell growth are associated with differential abilities to modulate specific PKC isoenzymes. PKC isozyme profiles were studied by Western-blot analysis in the cytosol, particulate and nuclear fractions of A549 and MCF-7 cells. PKCs-α, ?? and ?ζ were detected in both cell types with predominant location in the cytosol. Separation of cytosolic PKC isoenzymes in A549 cells by hydroxylapatite column chromatography and determination of PKC activity in fractions yielded a major peak which contained PKC-α. Exposure of cells to bryostatin I or TPA for 30 min caused the redistribution of PKCs-α and ?? from the cytosol to the particulate and nuclear fractions in a concentration-dependent fashion. PKC ?? was completely down-regulated by exposure to 10 nM bryostatin I for 18 hr or to TPA for 24 hr. Down-regulation of PKC-α was partial at 10 nM and complete at I μM of either agent. Bryostatin I inhibited incorporation of [³H]-labelled thymidine into cells only transiently, whereas TPA arrested growth for several days in A549 cells and irreversibly in MCF-7 cells. A549 cells, in which PKC was depleted by exposure to phorbol ester for 9 weeks, were resistant towards bryostatin-induced inhibition of DNA synthesis. The results suggest that the susceptibility of adenocarcinpma cells towards bryostatin-induced growth delay are determined by cellular levels of PKCs-α and/or ??. However, differences between bryostatin I and TPA in their abilities to inhibit cell growth do not seem to be intrinsically related to differences in redistribution or down-regulation of specific PKC isoenzymes.     

Effect of serum starvation on expression and phosphorylation of PKC-α and p53 in V79 cells: Implications for cell death

The effect of serum starvation on the expression and phosphorylation of PKC-α and p53 in Chinese hamster V79 cells was investigated. Serum starvation led to growth arrest, rounding up of cells and the appearance of new PKC-α and p53 bands on Western blots. Prolonged incubation (≥48 hr) in serum-deprived medium led to cell detachment and death. Moving cells to fresh medium containing 10% serum before, but not after, cell detachment reversed the changes observed in PKC-α and p53, and also prevented later cell detachment. Radiolabelling studies showed that the higher-molecular-weight PKC-α and p53 bands result from increased phosphorylation, while a lower-molecular-weight PKC-α band reflects newly synthesized protein. Immunocomplex kinase assays have shown that the increased phosphorylation of PKC-α is associated with its increased activity. To study the relationship between PKC-α, p53 and cell death, cells were treated either with TPA, to down-regulate PKC or with staurosporine, to inhibit PKC activity. Staurosporine, a potent PKC inhibitor and inducer of programmed cell death, caused the appearance of new PKC-α and p53 bands similar to those induced by serum starvation. If serum starvation was preceded by prolonged (48 hr) TPA treatment to down-regulate PKC-α, cell detachment and death did not take place within the same time frame. Intracellular fractionation of cells demonstrated that increased expression of PKC-α and the appearance of the associated higher and lower molecular-weight bands occurred in the nucleus. These data highlight the association of PKC-α and p53 with cellular events leading to cell death. Int. J. Cancer 80:400–405, 1999. © 1999 Wiley-Liss, Inc.     

Activation of protein kinase C prevents induction of apoptosis by geranylgeraniol in human leukemia HL60 cells

In a previous study, we showed that geranylgeraniol (GGO) is a potent inducer of apoptosis in human leukemia cells, including HL60 promyelocytic leukemia cells. The present study describes the effects of activators of protein kinase C (PKC) on GGO-induced apoptosis in various lines of leukemia cells. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and diacylglycerol (DG) inhibited the GGO-induced morphological changes that are characteristic of apoptosis and the DNA fragmentation. Similar effects were observed with other lines of human and murine leukemia cells such as MLI, U937, MI and P388. Flow cytometric analysis also revealed that both TPA and DG prevented GGO-induced DNA degradation in a dose-dependent manner. These inhibitory effects of TPA and DG were antagonized by inhibitors of PKC such as H-7 and staurosporin, and by amiloride, an inhibitor of Na⁺/H⁺ antiporter. In contrast to the inhibitory effects of TPA and DG on GGO-induced apoptosis, 4α-TPA, which is unable to activate PKC, failed to prevent GGO-induced DNA fragmentation. However, the selective activator of PKC-β, 12-deoxyphorbol 13-phenylacetate 20-acetate, significantly inhibited GGO-induced DNA fragmentation. Our results suggest that PKC, and in particular the PKC-β isoenzyme, might be involved in the process of GGO-induced apoptosis. Int. J. Cancer 71:691-697, 1997. © 1997 Wiley-Liss, Inc.     

Induction of CD44 expression by the epstein-barr virus latent membrane protein LMP1 is associated with lymphoma dissemination

Epstein-Barr Virus (EBV) is implicated in the pathogenesis of endemic Burkitt's lymphoma (BL), B-cell lymphomas occuring under immunosuppression, nasopharyngeal carcinoma and Hodgkin's disease. Two distinct patterns of latent EBV gene expression occur in EBV-associated lymphomas. BLs typically display expression of the nuclear antigen EBNAI only, whereas EBV-associated, non-Burkitt B-cell lymphomas express at least 9 latent viral genes (6 EBNAs and 3 latent membrane proteins), reminiscent of in vitro EBV-immortalized lymphoblastoid cell lines (LCL). BLs are characterized by local, extra-nodal growth, whereas EBV-associated B-cell lymphomas often disseminate to peripheral lymphoid tissue. We show here that BL cells forming local tumors after xenotransplantation into SCID mice disseminate to lymphoid tissue following introduction of the latent membrane protein 1 (LMP1) gene. Introduction of LMP1 into BL cells induced expression of CD44 on the cell surface, a molecule implicated in enhanced lymphoid tumor growth and dissemination. Introduction of CD44 into LMP1-/CD44-BL cells was observed to confer the disseminated tumor growth pattern associated with LMP1 expression. Taken together our results show that expression of LMP1 may regulate expression of CD44 and play an important role in the behavior of EBV-based lymphomas. © 1995 Wiley-Liss, Inc.     

Protein kinase C�� expression in breast cancer as measured by real-time PCR, western blotting and ELISA

The protein kinase C (PKC) family of genes encode serine/threonine kinases that regulate proliferation, apoptosis, cell survival and migration. Multiple isoforms of PKC have been described, one of which is PKCδ. Currently, it is unclear whether PKCδ is involved in promoting or inhibiting cancer formation/progression. The aim of this study was therefore to investigate the expression of PKCδ in human breast cancer and relate its levels to multiple parameters of tumour progression. Protein kinase Cδ expression at the mRNA level was measured using real-time PCR (n=208) and at protein level by both immunoblotting (n=94) and ELISA (n=98). Following immunoblotting, two proteins were identified, migrating with molecular masses of 78 and 160 kDa. The 78 kDa protein is likely to be the mature form of PKCδ but the identity of the 160 kDa form is unknown. Levels of both these proteins correlated weakly but significantly with PKCδ concentrations determined by ELISA (for the 78 kDa form, r=0.444, P<0.005, n=91 and for the 160 kDa form, r=0.237, P=0.023, n=91) and with PKCδ mRNA levels (for the 78 kDa form, r=0.351, P=0.001, n=94 and for the 160 kDa form, r=0.216, P=0.037, n=94). Protein kinase Cδ mRNA expression was significantly higher in oestrogen receptor (ER)-positive compared with ER-negative tumours (P=0.007, Mann–Whitney U-test). Increasing concentrations of PKCδ mRNA were associated with reduced overall patient survival (P=0.004). Our results are consistent with a role for PKCδ in breast cancer progression.     

Human prostate cell lines from normal and tumourigenic epithelia differ in the pattern and control of choline lipid headgroups released into the medium on stimulation of protein kinase C

Background:

Expression of protein kinase C alpha (PKCα) is elevated in prostate cancer (PCa); thus, we have studied whether the development of tumourigenesis in prostate epithelial cell lines modifies the normal pattern of choline (Cho) metabolite release on PKC activation.

Methods:

Normal and tumourigenic human prostate epithelial cell lines were incubated with [³H]-Cho to label choline phospholipids. Protein kinase C was activated with phorbol ester and blocked with inhibitors. Choline metabolites were resolved by ion-exchange chromatography. Phospholipase D (PLD) activity was measured by transphosphatidylation. Protein expression was detected by western blotting and/or RT–PCR. Choline uptake was measured on cells in monolayers over 60 min.

Results:

Normal prostate epithelial cell lines principally released phosphocholine (PCho) in contrast to tumourigenic lines, which released Cho. In addition, only with normal cell lines did PKC activation stimulate Cho metabolite release. Protein kinase C alpha expression varied between normal and tumourigenic cell lines but all showed a PKCα link to myristoylated alanine-rich C kinase substrate (MARCKS) protein. The five cell lines differed in Cho uptake levels, with normal PNT2C2 line cells showing highest uptake over 60 min incubation. Normal and tumourigenic cell lines expressed mRNA for PLD1 and PLD2, and showed similar levels of basal and PKC-activated PLD activity.

Conclusions:

The transition to tumourigenesis in prostate epithelial cell lines results in major changes to Cho metabolite release into the medium and PKC signalling to phosphatidylcholine turnover. The changes, which reflect the metabolic and proliferative needs of tumourigenic cells compared with untransformed cells, could be significant for both diagnosis and treatment.