Mildred Acevedo-Duncan

Estrogen stimulation of ovarian surface epithelial cell proliferation.

SummaryOvarian cancer is the leading cause of gynecological cancer mortality, and 85–90% of this malignancy originates from the ovarian surface epithelium (OSE). The etiology of ovarian epithelial cancer is unknown but a role for estrogens has been suspected. However, the effect of estrogens on OSE cell proliferation remains to be determined. Using the rabbit model, our studies have demonstrated that 17β-estradiol stimulates OSE cell proliferation and the formation of a papillary ovarian surface morphology similar to that seen in human ovarian serous neoplasms of low malignant potential. Immunohistochemical staining of ovarian tissue sections with an antibody to the estrogen receptor α demonstrates its expression in both OSE cells and stromal interstitial cells. In primary ovarian cell cultures, the proliferative response of the epithelial cells to 17β-estradiol depends on the expression of the estrogen receptor α in the epithelial cells. However, when the epithelial cells are grown together with ovarian stromal cells, their proliferative response to this hormone is greatly enhanced, suggesting the involvement of stromal-epithelial interactions. These studies suggest a role for estrogens and the estrogen receptor α in OSE growth.

Involvement of PKC-ι in glioma proliferation

Abstract.  Atypical protein kinase C‐iota (PKC‐ι) protects cells against apoptosis and may play a role in cell proliferation. However, in vivo, the status and function of PKC‐ι in human normal brain tissue, gliomas, benign and malignant meningiomas as well as its in vitro status in proliferating and confluent glioma cells, remains unknown. Objectives: The objectives of our research were to determine whether expression of PKC‐ι is altered either in gliomas or in benign and malignant meningiomas, compared to normal brain. In addition, we wished to establish the expression of PKC‐ι in proliferating plus in cell cycle‐arrested glioma cell lines, as well as the relationship between PKC‐ι siRNA on PKC‐ι protein content and cell proliferation. Materials and Methods: Western blot analyses for PKC‐ι were performed on 12 normal brain biopsies, 15 benign meningiomas, three malignant meningiomas and three gliomas. Results: Results demonstrated no (n = 9) or very weak (n = 3) detection of PKC‐ι in normal brain tissue. In comparison, PKC‐ι was robustly present in the majority of the benign meningiomas. Similarly, PKC‐ι was abundant in all malignant meningiomas and gliomas. Western blotting for PKC‐ι in confluent or proliferating glioma cell lines depicted substantial quantities of PKC‐ι in proliferating T98G and U‐138MG glioma cells. In contrast, confluent cells had either 71% (T98G) or 21% (U‐138MG) less PKC‐ι than proliferating cells. T98 and U‐138 MG glioma cells treated with 100 nm PKC‐ι siRNA had lower levels of cell proliferation compared to control siRNA‐A and complete down‐regulation of PKC‐ι protein content. Conclusion: These results support the concept that presence of PKC‐ι may be required for cell proliferation to take place.

Immunological evidence that insulin activates protein kinase C in BC3H-1 myocytes

Effects of insulin on immunoreactive protein kinase C were examined in BC3H‐1 myocytes. Insulin provoked rapid dose‐dependent decreases in cytosolic enzyme, and transient increases and subsequent decreases in membrane‐associated enzyme. Phorbol esters provoked similar changes. Our findings suggest that insulin provokes the translocative activation of protein kinase C.

A novel PKC-ι inhibitor abrogates cell proliferation and induces apoptosis in neuroblastoma

Protein Kinase C-iota (PKC-ι), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-ι confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of this research was to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-ι inhibitor in neuroblastoma cell proliferation and apoptosis. ICA-1 specifically inhibits the activity of PKC-ι but not that of PKC-zeta (PKC-ζ), the closely related atypical PKC family member. The IC(50) for the kinase activity assay was approximately 0.1μM which is 1000 times less than that of aurothiomalate, a known PKC-ι inhibitor. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-ι is an in vitro Cdk7 kinase and the phosphorylation of Cdk7 by PKC-ι was potently inhibited by ICA-1. Furthermore, our data shows that neuroblastoma cells proliferate via a PKC-ι/Cdk7/cdk2 cell signaling pathway and ICA-1 mediates its antiproliferative effects by inhibiting this pathway. ICA-1 (0.1μM) inhibited the in vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-ι inhibitor and chemotherapeutic agent for neuroblastoma.

Atypical protein kinase C phosphorylates IKKαβ in transformed non-malignant and malignant prostate cell survival

Mechanistic pathways involving atypical protein kinase C-iota (aPKC-iota) have been targeted in various cancer cells such as lung cancer, brain and prostate due to PKCiotas antiapoptotic function, and role in cell proliferation and cell survival. In the current study, we examined the involvement of PKC-iota in the NF-kappaB pathway following treatment of prostate cells with the pro-inflammatory cytokine tumor necrosis factor alpha (TNFalpha). Results demonstrated that androgen-independent DU-145 prostate carcinoma is insensitive to TNFalpha while transformed non-tumorigenic prostate RWPE-1 cells showed a slight sensitivity to TNFalpha. However, androgen-dependent LNCaP prostate cells are more sensitive to TNFalpha treatment and undergo apoptosis. Results demonstrated that in DU-145 cells, TNFalpha-induced PKC-iota in phosphorylation of IKKalphabeta. In RWPE-1 cells, PKC-zeta phosphorylates IKKalphabeta. Degradation of IkappaBalpha was observed in all three cell lines, allowing NF-kappaB/p65 translocation to the nucleus. Although, IKKalpha is weakly activated in LNCaP cells, the upstream kinase phosphorylation of IKKalphabeta via aPKCs was not observed. Hence, aPKCs may play a role in activation of NFkappaB pathway in prostate cancer cells.

Correction of metabolic abnormalities in a rodent model of obesity, metabolic syndrome, and type 2 diabetes mellitus by inhibitors of hepatic protein kinase C-ι

Excessive activity of hepatic atypical protein kinase (aPKC) is proposed to play a critical role in mediating lipid and carbohydrate abnormalities in obesity, the metabolic syndrome, and type 2 diabetes mellitus. In previous studies of rodent models of obesity and type 2 diabetes mellitus, adenoviral-mediated expression of kinase-inactive aPKC rapidly reversed or markedly improved most if not all metabolic abnormalities. Here, we examined effects of 2 newly developed small-molecule PKC-ι/λ inhibitors. We used the mouse model of heterozygous muscle-specific knockout of PKC-λ, in which partial deficiency of muscle PKC-λ impairs glucose transport in muscle and thereby causes glucose intolerance and hyperinsulinemia, which, via hepatic aPKC activation, leads to abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. One inhibitor, 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)], binds to the substrate-binding site of PKC-λ/ι, but not other PKCs. The other inhibitor, aurothiomalate, binds to cysteine residues in the PB1-binding domains of aPKC-λ/ι/ζ and inhibits scaffolding. Treatment with either inhibitor for 7 days inhibited aPKC, but not Akt, in liver and concomitantly improved insulin signaling to Akt and aPKC in muscle and adipocytes. Moreover, both inhibitors diminished excessive expression of hepatic, aPKC-dependent lipogenic, proinflammatory, and gluconeogenic factors; and this was accompanied by reversal or marked improvements in hyperglycemia, hyperinsulinemia, abdominal obesity, hepatosteatosis, hypertriglyceridemia, and hypercholesterolemia. Our findings highlight the pathogenetic importance of insulin signaling to hepatic PKC-ι in obesity, the metabolic syndrome, and type 2 diabetes mellitus and suggest that 1H-imidazole-4-carboxamide, 5-amino-1-[2,3-dihydroxy-4-[(phosphonooxy)methyl]cyclopentyl-[1R-(1a,2b,3b,4a)] and aurothiomalate or similar agents that selectively inhibit hepatic aPKC may be useful treatments.

Human glioma PKC‐ι and PKC‐βII phosphorylate cyclin‐dependent kinase activating kinase during the cell cycle

Cell cycle phase transition is regulated in part by the trimeric enzyme, cyclin‐dependent kinase activating kinase (CAK) which phosphorylates and activates cyclin‐dependent kinases (cdks). Protein kinase C (PKC) inhibitors prevent cell cycle phase transition, suggesting a fundamental role for PKCs in cell cycle regulation. We report that in glioma cells, CAK (cdk7) is constitutively associated with PKC‐ι. In vitro phosphorylation, co‐immunoprecipitation, and analysis of phosphorylated proteins by autoradiography indicate that CAK (cdk7) is a substrate for PKC‐ι and PKC‐βII hyperphosphorylation. These results establish a role for PKC‐ι and PKC‐βII in the activation of CAK during the glioma cell cycle.

Akt-Dependent Phosphorylation of Hepatic FoxO1 Is Compartmentalized on a WD40/ProF Scaffold and Is Selectively Inhibited by aPKC in Early Phases of Diet-Induced Obesity

Initiating mechanisms that impair gluconeogenic enzymes and spare lipogenic enzymes in diet-induced obesity (DIO) are obscure. Here, we examined insulin signaling to Akt and atypical protein kinase C (aPKC) in liver and muscle and hepatic enzyme expression in mice consuming a moderate high-fat (HF) diet. In HF diet–fed mice, resting/basal and insulin-stimulated Akt and aPKC activities were diminished in muscle, but in liver, these activities were elevated basally and were increased by insulin to normal levels. Despite elevated hepatic Akt activity, FoxO1 phosphorylation, which diminishes gluconeogenesis, was impaired; in contrast, Akt-dependent phosphorylation of glycogenic GSK3β and lipogenic mTOR was elevated. Diminished Akt-dependent FoxO1 phosphorylation was associated with reduced Akt activity associated with scaffold protein WD40/Propeller/FYVE (WD40/ProF), which reportedly facilitates FoxO1 phosphorylation. In contrast, aPKC activity associated with WD40/ProF was increased. Moreover, inhibition of hepatic aPKC reduced its association with WD40/ProF, restored WD40/ProF-associated Akt activity, restored FoxO1 phosphorylation, and corrected excessive expression of hepatic gluconeogenic and lipogenic enzymes. Additionally, Akt and aPKC activities in muscle improved, as did glucose intolerance, weight gain, hepatosteatosis, and hyperlipidemia. We conclude that Akt-dependent FoxO1 phosphorylation occurs on the WD/Propeller/FYVE scaffold in liver and is selectively inhibited in early DIO by diet-induced increases in activity of cocompartmentalized aPKC.

Biopathology of Ovarian Mesothelium

The ovarian surface epithelium, formerly called germinal epithelium, is a modified mesothelium, which covers the peritoneal aspect of the mammalian ovary [1]. During the last decade, this tissue has received increasing attention due to its putative role in ovulation and in gynecological oncology as the source of a most frequent and often lethal form of human ovarian cancer [1]. The morphological changes undergone by the ovarian mesothelium (OM) during the prepuberal, reproductive, and postmenopausal years have been already discussed in previous reviews (2, 3). This chapter will address recent and relevant research on developmental aspects of the OM and will attempt to highlight gaps and future investigative directions in the biopathology of this tissue. To this end, the following topics will be covered: morphological changes of OM throughout life, ultrastructure and immunocytochemistry of OM, comparative biology of OM, physiopatho-logy of OM, in vitro OM models, rabbit experimental OM model, pathobiology of ovarian and extraovarian mesothelia, and new frontiers in OM research.

PKC-ι promotes glioblastoma cell survival by phosphorylating and inhibiting BAD through a phosphatidylinositol 3-kinase pathway.

The focus of this research was to investigate the role of protein kinase C-iota (PKC-ι) in regulation of Bad, a pro-apoptotic BH3-only molecule of the Bcl-2 family in glioblastoma. Robust expression of PKC-ι is a hallmark of human glioma and benign and malignant meningiomas. The results were obtained from the two human glial tumor derived cell lines, T98G and U87MG. In these cells, PKC-ι co-localized and directly associated with Bad, as shown by immunofluorescence, immunoprecipitation, and Western blotting. Furthermore, in-vitro kinase activity assay showed that PKC-ι directly phosphorylated Bad at phospho specific residues, Ser-112, Ser-136 and Ser-155 which in turn induced inactivation of Bad and disruption of Bad/Bcl-XL dimer. Knockdown of PKC-ι by siRNA exhibited a corresponding reduction in Bad phosphorylation suggesting that PKC-ι may be a Bad kinase. PKC-ι knockdown also induced apoptosis in both the cell lines. Since, PKC-ι is an essential downstream mediator of the PI (3)-kinase, we hypothesize that glioma cell survival is mediated via a PI (3)-kinase/PDK1/PKC-ι/Bad pathway. Treatment with PI (3)-kinase inhibitors Wortmannin and LY294002, as well as PDK1 siRNA, inhibited PKC-ι activity and subsequent phosphorylation of Bad suggesting that PKC-ι regulates the activity of Bad in a PI (3)-kinase dependent manner. Thus, our data suggest that glioma cell survival occurs through a novel PI (3)-kinase/PDK1/PKC-ι/BAD mediated pathway.