Peter Acs

NON-EQUIVALENT ROLES FOR THE FIRST AND SECOND ZINC FINGERS OF PROTEIN KINASE CDELTA : EFFECT OF THEIR MUTATION ON PHORBOL ESTER-INDUCED TRANSLOCATION IN NIH 3T3 CELLS

Classical and novel protein kinase C (PKC) isozymes contain two, so-called cysteine-rich zinc finger domains that represent the binding sites for phorbol esters and the diacylglycerols. X-ray crystallographic, mutational, and modeling studies are providing detailed understanding of the interactions between the phorbol esters and individual PKC zinc fingers. In the present study, we explore the roles of the individual zinc fingers in the context of the intact enzyme. Our approach was to mutate either the first, the second, or both zinc fingers of PKCδ, to express the mutated enzyme in NIH 3T3 cells, and to monitor the effect of the mutations on the dose-response curve for translocation induced by phorbol 12-myristate 13-acetate. The introduced mutations change into glycine the consensus proline in the phorbol ester binding loop of the zinc finger; in the isolated zinc finger, this mutation causes a 125-fold decrease in phorbol ester binding affinity. We observed that mutation in the first zinc finger caused almost no shift in the dose-response curve for translocation; mutation in the second zinc finger caused a 21-fold shift, whereas mutation in both zinc fingers caused a 138-fold shift. We conclude that the zinc fingers in the intact PKC are not equivalent and that the second zinc finger plays the predominant role in translocation of protein kinase Cδ in response to phorbol 12-myristate 13-acetate. Our findings have important implications for the understanding and design of PKC inhibitors targeted to the zinc finger domains.

Specific vanilloid responses in C6 rat glioma cells

Capsaicin and its ultrapotent analog resiniferatoxin (RTX) act through specific vanilloid receptors on sensory neurons. Here, we describe specific vanilloid responses in rat C6 glioma cells. Capsaicin and RTX stimulated 45Ca uptake in a similar fashion to that found for cultured rat dorsal root ganglion neurons (DRGs); this response was antagonized by the antagonists capsazepine and ruthenium red. As in DRGs, pretreatment of C6 cells with capsaicin or RTX produced desensitization to subsequent stimulation of 45Ca uptake. The potency for desensitization by RTX in the C6 cells corresponded to that for 45Ca uptake, whereas in DRGs it occurred at significantly lower concentrations corresponding to that for the high affinity [3H]RTX binding site. Consistent with this difference, in C6 cells we were unable to detect [3H]RTX binding. These characteristics suggest the presence of C-type but not R-type vanilloid receptors on C6 cells. After 2 day treatment, capsaicin but not RTX inhibited the proliferation and altered the differentiation of the cells and produced apoptosis. In the long term experiments, capsazepine, instead of antagonizing the effect of capsaicin, acted as an agonist. Moreover, capsazepine displayed these effects with higher potency than that of capsaicin. The different potencies and structure activity relations suggest a distinct mechanism for these long-term vanilloid effects. Our finding that C6 cells can respond directly to capsaicin necessitates a reevaluation of the in vivo pathway of response to vanilloids, and highlights the importance of the neuron-glial network.

The Effect of Oncotype DX Recurrence Score on Treatment Recommendations for Patients with Estrogen Receptor–Positive Early Stage Breast Cancer and Correlation with Estimation of Recurrence Risk by Breast Cancer Specialists

PURPOSE The Oncotype DX assay predicts likelihood of distant recurrence and improves patient selection for adjuvant chemotherapy in estrogen receptor-positive (ER-positive) early stage breast cancer. This study has two primary endpoints: to evaluate the impact of Oncotype DX recurrence scores (RS) on chemotherapy recommendations and to compare the estimated recurrence risk predicted by breast oncology specialists to RS. METHODS One hundred fifty-four patients with ER-positive early stage breast cancer and available RS results were selected. Clinicopathologic data were provided to four surgeons, four medical oncologists, and four pathologists. Participants were asked to estimate recurrence risk category and offer their chemotherapy recommendations initially without and later with knowledge of RS results. The three most important clinicopathologic features guiding their recommendations were requested. RESULTS Ninety-five (61.7%), 45 (29.2%), and 14 (9.1%) tumors were low, intermediate, and high risk by RS, respectively. RS significantly correlated with tumor grade, mitotic activity, lymphovascular invasion, hormone receptor, and HER2/neu status. Estimated recurrence risk by participants agreed with RS in 54.2% ± 2.3% of cases. Without and with knowledge of RS, 82.3% ± 1.3% and 69.0% ± 6.9% of patients may be overtreated, respectively (p = 0.0322). Inclusion of RS data resulted in a 24.9% change in treatment recommendations. There was no significant difference in recommendations between groups of participants. CONCLUSIONS Breast oncology specialists tended to overestimate the risk of tumor recurrence compared with RS. RS provides useful information that improves patient selection for chemotherapy and changes treatment recommendations in approximately 25% of cases.

Differential role of specific PKC isoforms in the proliferation of glial cells and the expression of the astrocytic markers GFAP and glutamine synthetase

In this study, we explored the role of specific protein kinase C (PKC) isoforms in glial cell proliferation and on the expression of the astrocytic markers GFAP and glutamine synthetase using C6 cells as a model. Analysis of the expression of the various PKC isoforms in control and differentiated C6 cells revealed differences in the expression of specific PKC isoforms. Undifferentiated C6 cells, which express low levels of GFAP and glutamine synthetase (GS), have high levels of PKCalpha and delta, whereas differentiated C6 cells, which express higher levels of both GFAP and GS have lower levels of PKCalpha and delta and higher levels of PKCgamma, theta and eta. Using C6 cells overexpressing specific PKC isoforms, we examined the role of these isoforms on the proliferation and differentiation of C6 cells. Cells overexpressing PKCalpha displayed a reduced level of GFAP, whereas GS expression was not affected. On the other hand, cells overexpressing PKCdelta showed reduced GS expression but little effect on GFAP. Finally, cells expressing PKCgamma displayed a marked increase in the levels of both GFAP and GS. The proliferation of C6 cells was increased in cells overexpressing PKCalpha and epsilon and decreased in cells overexpressing PKCgamma, delta and eta. The results of this study suggest that glial cell proliferation and astrocytic differentiation can be regulated by specific PKC isoforms that selectively affect cell proliferation and the expression of the two astrocytic markers GFAP and GS.

Opposite roles of protein kinase C isoforms in proliferation, differentiation, apoptosis, and tumorigenicity of human HaCaT keratinocytes

We have previously shown that the protein kinase C (PKC) system plays a pivotal role in regulation of proliferation and differentiation of the human keratinocyte line HaCaT which is often used to assess processes of immortalization, transformation, and tumorigenesis in human skin. In this paper, using pharmacological and molecular biology approaches, we investigated the isoform-specific roles of certain PKC isoenzymes (conventional cPKCα and β; novel nPKCδ and ε) in the regulation of various keratinocyte functions. cPKCα and nPKCδ stimulated cellular differentiation and increased susceptibility of cells to actions of inducers of apoptosis, and they markedly inhibited cellular proliferation and tumor growth in immunodeficient mice. In marked contrast, cPKCβ and nPKCε increased both in vitro and in vivo growth of cells and inhibited differentiation and apoptosis. Our data present clear evidence for the specific, antagonistic roles of certain cPKC and nPKC isoforms in regulating the above processes in human HaCaT keratinocytes.

Protein Kinase C δ (PKCδ) Inhibits the Expression of Glutamine Synthetase in Glial Cells via the PKCδ Regulatory Domain and Its Tyrosine Phosphorylation

Protein kinase C (PKC) plays an important role in the proliferation and differentiation of glial cells. In a recent study we found that overexpression of PKCδ reduced the expression of the astrocytic marker glutamine synthetase (GS). In this study we explored the mechanisms involved in the inhibitory effect of PKCδ on the expression of glutamine synthetase. Using PKC chimeras we first examined the role of the catalytic and regulatory domains of PKCδ on the expression of glutamine synthetase. We found that cells stably transfected with chimeras between the regulatory domain of PKCδ and the catalytic domains of PKCα or ε inhibited the expression of GS, similar to the inhibition exerted by overexpression of PKCδ itself. In contrast, no significant effects were observed in cells transfected with the reciprocal PKC chimeras between the regulatory domains of PKCα or ε and the catalytic domain of PKCδ. PKCδ has been shown to undergo tyrosine phosphorylation in response to various activators. Tyrosine phosphorylation of PKCδ in response to phorbol 12-myristate 13-acetate and platelet-derived growth factor occurred only in chimeras which contained the PKCδ regulatory domain. Cells transfected with a PKCδ mutant (PKCδ5), in which the five putative tyrosine phosphorylation sites were mutated to phenylalanine, showed markedly diminished tyrosine phosphorylation in response to phorbol 12-myristate 13-acetate and platelet-derived growth factor and normal levels of GS. Our results indicate that the regulatory domain of PKCδ mediates the inhibitory effect of this isoform on the expression of GS. Phosphorylation of PKCδ on tyrosine residues in the regulatory domain is implicated in this inhibitory effect.

Regulation of actin cytoskeleton in lymphocytes: PKC‐δ disrupts IL‐3–induced membrane ruffles downstream of Rac1

In the murine pre‐B lymphoid cell line Baf3, the presence of IL‐3 is required for the formation of membrane ruffles that intensely stain for actin and are responsible for the elongated cell phenotype. Withdrawal of IL‐3 dissolves ruffled protrusions and converts the cell phenotype to round. Flow cytometric analysis of the cell shape showed that an inactive analog of Rac1 but not inactive RhoA or inactive cdc42 rounds the cells in the presence of IL‐3. Constitutively activated Rac1 restores the elongated cell phenotype to IL‐3–starved cells. We conclude that the activity of Rac1 is necessary and sufficient for the IL‐3–induced assembly of membrane ruffles. Similar to the IL‐3 withdrawal, phorbol 12‐myristate 13‐acetate (PMA) dissolves actin‐formed membrane ruffles and rounds the cells in the presence of IL‐3. Flow cytometric analysis of the cell shape demonstrated that in the presence of IL‐3 the PMA‐induced cell rounding cannot be abolished by constitutively active Rac1 but can be imitated by inactive Rac1. These data indicate that PMA disrupts the IL‐3 pathway downstream of Rac1. Cells rounded by PMA return to the elongated phenotype concomitantly with PKC depletion. PMA‐induced cell rounding can be reversed by the PKC‐specific inhibitor GF109203X. Experiments with overexpression in Baf3 of individual PKC isoforms and a dominant negative PKC‐δ indicate that activation of PKC‐δ but not other PKC isoforms is responsible for disruption of membrane ruffles. J. Cell. Physiol. 179:157–169, 1999. Published 1999 Wiley‐Liss, Inc.

Inhibitors of Protein Kinase C and Related Receptors for the Lipophilic Second-Messenger sn-1,2-Diacylglycerol

Toxic natural products, selected by evolution both for activity against crucial biological targets and for potency, have made major contributions in highlighting such targets and in defining their functions. The phorbol esters, initially isolated on the basis of their activity as tumor promoters and as acute irritants, were found to have dramatic biological effects in virtually every system in which they were examined (1). We now appreciate that the phorbol esters function as ultrapotent analogs of the lipophilic second messenger sn-1,2-diacylglycerol (DAG) and that phorbol ester receptors identify high-affinity targets in DAG signaling (2), of which protein kinase C (PKC) has been most thoroughly studied (3–7).

Estimation of Risk of Recurrence of Early Stage Estrogen Receptor Positive Breast Carcinoma by Surgical and Medical Oncologists and Pathologists Compared to the Oncotype Dx® Recurrence Score.

Background: The decision to use adjuvant chemotherapy in patients with early stage breast cancer is based in part on the estimation of risk of tumor recurrence by physicians, which traditionally relies heavily on tumor size, nodal status and a set of biologic tumor characteristics such as hormone receptor and HER2 expression. The Oncotype DX® assay is a 21-gene expression profile aiming to improve risk stratification, recurrence prediction and optimize selection of patients for adjuvant chemotherapy.Methods: We selected 154 consecutive patients with early stage estrogen receptor (ER) positive breast cancer and available Oncotype Dx® recurrence score (RS) for the study. Clinicopathologic data, including patient age, menopausal status, tumor size, histologic type, grade, mitotic activity, presence of lymphatic invasion (LVI), nodal status, hormone receptor and HER2 status on all patients were provided to four surgical oncologists, four medical oncologists and three pathologists, specializing in breast cancer diagnosis and management. Participants were asked to estimate the risk of recurrence of tumors based on available clinicopathologic data and to provide the three most important tumor features their risk estimates were based on. Risk estimates of participants were compared with RS results.Results: Based on the Oncotype Dx® results, 95 (61.7%), 45 (29.2%) and 14 (9.1%) tumors were of low (RS Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4061.