I. Bernard Weinstein

Overproduction of protein kinase C causesdisordered growth control in rat fibroblasts

We have generated a series of rat fibroblast cell lines that stably overexpress a full-length cDNA encoding the beta 1 form of protein kinase C (PKC). These cell lines contain a 20- to 53-fold increase in PKC activity and exhibit dramatically enhanced morphologic changes following exposure to the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). They grow to a high saturation density in monolayer cultures and, when maintained at postconfluence, develop small, dense foci. In contrast to control cells, which display complete anchorage dependence, PKC-overproducing cells form small colonies in soft agar in the absence of TPA and large colonies in the presence of TPA. Thus, the mere overproduction of a single form of PKC is sufficient to confer multiple growth abnormalities in rat fibroblasts. These results provide direct evidence that PKC plays a critical role in growth control and that it mediates several of the cellular effects of the phorbol ester tumor promoters. They also suggest that the activation of PKC may be of central importance in the process of multistage carcinogenesis.

Multiple functions of p27Kip1 and its alterations in tumor cells: a review

Cyclin‐dependent kinases (CDKs), together with cyclins, their regulatory subunits, govern cell‐cycle progression in eukaryotic cells. p27Kip1 is a member of a family of CDK inhibitors (CDIs) that bind to cyclin/CDK complexes and arrest cell division. There is considerable evidence that p27Kip1 plays an important role in multiple fundamental cellular processes, including cell proliferation, cell differentiation, and apoptosis. Moreover, p27Kip1 is a putative tumor‐suppressor gene that appears to play a critical role in the pathogenesis of several human malignancies and its reduced expression has been shown to correlate with poor prognosis in cancer patients. This study reviews current information on the functions of p27Kip1, its abnormalities found in human tumors, and the possible clinical implications of these findings with respect to the management of cancer patients. J. Cell. Physiol. 183:18–27, 2000.

(−)-Epigallocatechin Gallate and Polyphenon E Inhibit Growth and Activation of the Epidermal Growth Factor Receptor and Human Epidermal Growth Factor Receptor-2 Signaling Pathways in Human Colon Cancer Cells

Purpose: (−)-Epigallocatechin gallate (EGCG) inhibits activation of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2) and multiple downstream signaling pathways in cancer cell lines. In this study we compared the cellular and molecular effects of EGCG with a well-standardized decaffeinated green tea catechin mixture Polyphenon E (Poly E) on human colon cancer cell lines. Experimental Design and Results: Both EGCG and Poly E preferentially inhibited growth of the Caco2, HCT116, HT29, SW480, and SW837 colon cancer cells when compared with the FHC normal human fetal colon cell line. The EGFR and HER2 proteins were overexpressed and constitutively activated in all of the colon cancer cell lines when compared with the FHC cell line. Treatment of HT29 cells with EGCG or Poly E caused an increase of cells in G1 and induced apoptosis. Both EGCG and Poly E caused a decrease in the phosphorylated forms of EGFR and HER2 proteins, and subsequently caused a decrease in the phosphorylated forms of the extracellular signal-regulated kinase and Akt proteins. Similar effects of these compounds were seen when the cells were stimulated with transforming growth factor α. Reporter assays indicated that both EGCG and Poly E inhibited the transcriptional activity of the activator protein 1 (AP-1), c-fos, nuclear factor κB, and cyclin D1 promoters. The combination of only 1 μg/mL of epicatechin plus 10 μg/mL of EGCG displayed synergistic effects on growth inhibition and induction of apoptosis. Furthermore, when treatment was prolonged for 96 hours, 1 μg/mL of EGCG or Poly E was sufficient to inhibit growth, reduce activation of EGFR and HER2, and induce apoptosis. Conclusion: Our findings suggest that EGCG or Poly E may be useful in the chemoprevention and/or treatment of colon cancer. Poly E contains about 60% EGCG, yet pure EGCG and Poly E had similar potencies (expressed as μg/ml). Poly E may be preferable because it is easier to prepare and this mixture of catechins may exert synergistic effects.

Roles of Specific Isoforms of Protein Kinase C in the Transcriptional Control of Cyclin D1 and Related Genes

Although protein kinase C (PKC) has been implicated in cell cycle progression, cell proliferation, and tumor promotion, the precise roles of specific isoforms in these processes is not clear. Therefore, we constructed and analyzed a series of expression vectors that encode hemagglutinin-tagged wild type (WT), constitutively active mutants (ΔNPS and CAT), and dominant negative mutants of PKCs α, β1, β2, γ, δ, ϵ, η, ζ, and ι. Cyclin D1 promoter reporter assays done in serum-starved NIH3T3 cells indicated that the constitutively active mutants of PKC-α and PKC-ϵ were the most potent activators of this reporter, whereas the constitutively active mutant of PKC-δ inhibited its activity. Transient transfection studies with a series of 5′-deleted cyclin D1 promoter constructs showed that the proximal 964-base region, which contains AP-1, SP1, and CRE enhancer elements, is required for activation of the cyclin D1 promoter by PKC-α. Deletion of the AP-1 enhancer element located at position –954 upstream from the initiation site abolished PKC-α-dependent activation of cyclin D1 expression. Deletion of the SP1 or CRE enhancer elements did not have any effect. A dominant negative mutant of c-Jun inhibited activation of the cyclin D1 promoter in a concentration-dependent manner, providing further evidence that AP-1 activity is required for activation of the cyclin D1 promoter by PKC-α and PKC-ϵ. The constitutively active mutants of PKC-α and PKC-ϵ also activated c-fos, c-jun, and cyclin E promoter activity. Furthermore, NIH3T3 cells that stably express the constitutively active mutants of PKC-α or PKC-ϵ displayed increased expression of endogenous cyclins D1 and E and faster growth rates. These results provide evidence that the activation of PKC-α or PKC-ϵ in mouse fibroblasts can play an important role in enhancing cell cycle progression and cell proliferation.

A preparative method for obtaining enucleated mammalian cells

Summary Mouse L cells can be enucleated in suspension by centrifugation in discontinuous Ficoll density gradients while in the presence of Cytochalasin B. Greater than 50% of the cytoplasts thus obtained attach to glass or plastic and undergo morphologic recovery within 2–4 hours of replating. Protein synthesis in cytoplasts undergoes a biphasic decay from an initial rate of approximately 50% of control nucleated cells. This method can yield up to 5 × 108 cytoplasts with consistently low levels of contamination by nucleated cells (less than 0.2%), and is well suited for obtaining quantitative amounts of cytoplasts or karyoplasts for physiologic or biochemical studies.

Exisulind (sulindac sulfone) suppresses growth of human prostate cancer in a nude mouse xenograft model by increasing apoptosis

OBJECTIVES Recent studies have shown that Exisulind, a sulfone metabolite of the nonsteroidal anti-inflammatory drug (NSAID) sulindac, has inhibitory activity in vitro with cultured human prostate cancer cells. To determine whether this effect might be pharmacologically relevant in vivo, we tested whether Exisulind therapy could suppress the growth of human prostate cancer cells in a nude mouse xenograft model. METHODS Thirty athymic nude mice were injected subcutaneously in the flank with 1 x 10(7) LNCaP human prostate tumor cells. All mice received a control diet for 21 days. One group of mice was continued on this control diet for an additional 4 weeks, a second group was switched to a diet supplemented with 0.05% Exisulind (40% of maximal tolerated dose [MTD]), and a third group was switched to a diet supplemented with 0.1% Exisulind (80% MTD) for the additional 4 weeks. Tumor growth was measured through the 4-week test period, and subsequently tissue sections from the various groups were tested for apoptotic and dividing cells by quantified use of the TUNEL assay and a bromodeoxyuridine (BrdU) incorporation immunoassay. RESULTS Tumors grew by 158%, 24%, and 18% for the control and 0.05% and 0.1% Exisulind groups, respectively (P = 0.02) during the 4-week test period. Immunohistochemical studies on excised tumors showed an increased number of apoptotic bodies in the treated groups versus the control group (P<0.0001) but no change in the number of BrdU positive cells. CONCLUSIONS This is the first study to show a direct in vivo effect of an NSAID-derived drug, lacking cyclooxygenase inhibitory activity, in a xenograft model of prostate cancer. Clinical studies to evaluate the effects of Exisulind against prostate cancer in humans are warranted.

Deletion of histidine triad nucleotide-binding protein 1/PKC-interacting protein in mice enhances cell growth and carcinogenesis

PKC-interacting protein (PKCI), also designated histidine triad nucleotide-binding protein 1, belongs to the histidine triad (HIT) family of proteins. Its structure is highly conserved from bacteria to humans and shares homology with the tumor-suppressor gene fragile histidine triad (FHIT). Although it was originally thought to inhibit PKC, its actual physiologic function is not known. Therefore, we used the technique of homologous recombination to generate homozygous deleted PKCI-/- mice. These mice display normal fetal and adult development. However, when mouse embryo fibroblasts were established from 13.5-day embryos and serially passaged the PKCI-/- cells displayed an increase in growth rate and underwent spontaneous immortalization, whereas the PKCI+/+ cells senesced and ceased growing. Furthermore, the PKCI-/- mouse embryo fibroblasts displayed increased resistance to cytotoxicity by ionizing radiation. In view of these findings we examined possible effects of PKCI on susceptibility to carcinogenicity. Both PKCI+/+ and PKCI-/- mice were treated with the chemical carcinogen N-nitrosomethylbenzylamine (NMBA) by intragastric administration and killed 12 weeks later. As expected with this protocol, NMBA induced squamous tumors (both papillomas and carcinomas) of the forestomach. The incidence, multiplicity per mouse, volume, and degree of malignancy of these tumors were significantly greater in the PKCI-/- than in the PKCI+/+ mice. Furthermore, four adenomas and one adenocarcinoma of the glandular stomach were found in the NMBA-treated PKCI-/- mice but no tumors of the glandular stomach were found in the NMBA-treated PKCI+/+ mice or in any of the untreated mice. Taken together, these findings suggest that, like FHIT, PKCI may normally play a tumor-suppressor role. The possible role of PKCI as a tumor suppressor in humans remains to be determined.

Activation of Protein Kinase G Is Sufficient to Induce Apoptosis and Inhibit Cell Migration in Colon Cancer Cells

The activation of protein kinase G (PKG) by cGMP has become of considerable interest as a novel molecular mechanism for the induction of apoptosis in cancer cells, because sulindac sulfone (exisulind, Aptosyn) and certain derivatives that inhibit cGMP-phosphodiesterases and thereby increase cellular levels of cGMP appear to induce apoptosis via this mechanism. However, other effects of these compounds have not been excluded, and the precise mechanism by which PKG activation induces apoptosis has not been elucidated in detail. To directly examine the effects of PKG on cell growth and apoptosis, we generated a series of mutants of PKG Iα: PKG IαS65D, a constitutively activated point mutant; PKG IαΔ, a constitutively activated N-terminal truncated mutant; and PKG IαK390R, a dominant-negative point mutant. A similar series of mutants of PKG Iβ were also constructed (Deguchi et al., Mol. Cancer Ther., 1: 803–809, 2002). The present study demonstrates that when transiently expressed in SW480 colon cancer, the constitutively activated mutants of PKG Iβ, and to a lesser extent PKG Iα, inhibit colony formation and induce apoptosis. We were not able to obtain derivatives of SW480 cells that stably expressed these constitutively activated mutants, presumably because of toxicity. However, derivatives that stably overexpressed wild-type PKG Iβ displayed growth inhibition, whereas derivatives that stably expressed the dominant-negative mutant (KR) of PKG Iβ grew more rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG Iβ was associated with decreased cellular levels of β-catenin and cyclin D1 and increased levels of p21CIP1. Reporter assays indicated that activation of PKG Iβ inhibits the transcriptional activity of the cyclin D1 promoter. We also found that transient expression of the constitutively activated mutants of PKG Iβ inhibited cell migration. Taken together, these results indicate that activation of PKG Iβ is sufficient to inhibit growth and cell migration and induce apoptosis in human colon cancer cells and that these effects are associated with inhibition of the transcription of cyclin D1 and an increase in the expression of p21CIP1.

Regulation of Airway Epithelial Cell NF-κB-Dependent Gene Expression by Protein Kinase Cδ

Airway epithelial cells synthesize proinflammatory molecules such as IL-8, GM-CSF, RANTES, and ICAM-1, the expression of which is increased in the airways of patients with asthma. We investigated the regulation of these NF-κB-dependent genes by the novel protein kinase C (PKC) isoform PKCδ in 16HBE14o- human airway epithelial cells, focusing on IL-8 expression. Transient transfection with the constitutively active catalytic subunit of PKCδ (PKCδ-CAT), and treatment with bryostatin 1, an activator of PKCδ, each increased transcription from the IL-8 promoter, whereas overexpression of PKCε had minor effects. Expression of a dominant negative PKCδ mutant (PKCδ-KR) or pretreatment of cells with rottlerin, a chemical PKCδ inhibitor, attenuated TNF-α- and phorbol ester-induced transcription from the IL-8 promoter. Bryostatin 1 treatment increased IL-8 protein abundance in primary airway epithelial cells. Selective activation of PKCδ by bryostatin also activated NF-κB, as evidenced by p65 RelA and p50 NF-κB1 binding to DNA, NF-κB trans-activation, and IκB degradation. The sufficiency of PKCδ to induce NF-κB nuclear translocation and binding to DNA was confirmed in a 16HBE14o- cell line inducibly expressing PKCδ-CAT under the tet-off system. Deletion of the NF-κB response element severely attenuated PKCδ-induced IL-8 promoter activity. Finally, PKCδ-CAT induced transcription from the GM-CSF, RANTES, and ICAM-1 promoters. Together these data suggest that PKCδ plays a key role in the regulation of airway epithelial cell NF-κB-dependent gene expression.

Activation of rat brain protein kinase C by lipid oxidation products

The unsaturated fatty acid components of membrane lipids are susceptible to oxidation in vitro and in vivo. The initial oxidation products are hydroperoxy fatty acids that are converted spontaneously or enzymatically to a variety of products. Hydroperoxy derivatives of oleic, linoleic, or arachidonic acids stimulate the activity of protein kinase C (PKC) purified from rat brain. The hydroperoxy acids satisfy the requirement of PKC for phospholipid (e.g., phosphatidylserine). Activation is observed in the presence or absence of 1 mM Ca2+. Reduction of the hydroperoxides to alcohols or dehydration of the hydroperoxides to ketones increases the Ka for activation three- to fourfold but does not significantly reduce the maximal extent of PKC activation. The Kas for activation by hydroperoxy acids are approximately half the values exhibited by the unoxidized fatty acids. Since oxidation of unsaturated fatty acids to hydroperoxides is the first event in lipid peroxidation, activation of PKC by hydroperoxy fatty acids may be an early cellular response to oxidative stress.