The characterization of PKC-isozyme specific EGFR-dependent Erk1/2 activation in androgen-independent prostate cancer cell lines

One growth factor receptor commonly altered during prostate tumor progression is the epidermal growth factor receptor (EGFR). EGFR signaling regulates Erk1/2 phosphorylation through multiple mechanisms. We hypothesized that PKC isozymes play a role in EGFR-dependent signaling, and that through PKC isozyme selective inhibition, EGFR-dependent Erk1/2 activation can be attenuated in AICaP cells. ^ To test the hypothesis, PKC activation was induced by 12-O-tetradecanoyi-phorbol-13-acetate (TPA) in PC-3 cells. As a result, Erk1/2 was activated similarly to what was observed upon EGF stimulation. EGF-induced Erk1/2 activation in PC-3 cells was PKC-dependent, as demonstrated through use of a selective PKC inhibitor, GF109203X. This provides evidence for PKC regulatory control over Erk1/2 signaling downstream of EGFR. Next, we demonstrated that when PKC was inhibited by GF109203X, EGF-stimulated Erk1/2 activation was inhibited in PC-3, but not DU145 cells. TPA-stimulated Erk1/2 activation was EGFR-dependent in both DU145 and PC-3 cells, demonstrated through abrogation of Erk1/2 activation by a selective EGFR inhibitor AG1478. These data support PKC control at or upstream of EGFR in AICaP cells. We observed that interfering with ligand/EGFR binding abrogated Erk1/2 signaling in TPA-stimulated cells, revealing a role for PKC upstream of EGFR. ^ Next, we determined which PKC isozymes might be responsible for Erk1/2 regulation. We first determined that human AICaP cell lines express the same PKC isozymes as those observed in clinical prostate cancer specimens (α, ϵ, &zgr;, ι and PKD). Isozyme-selective methods were employed to characterize discrete PKC isozyme function in EGFR-dependent Erk1/2 activation. Pharmacologic inhibitors implicated PKCα in TPA-induced EGFR-dependent Erk1/2 activation in both PC-3 and DU145 cells. Further, the cPKC-specific inhibitor, Gö6976 decreased viablilty of DU145 cells, providing evidence that PKCα is necessary for growth and survival. Finally, resveratrol, a phytochemical with strong cancer therapeutic potential inhibited Erk1/2 activation, and this correlated with selective inhibition of PKCα. These results demonstrate that PKC regulates pathways critical to progression of CaP cells, including those mediated by EGFR. Thus, PKC isozyme-selective targeting is an attractive therapeutic strategy, and understanding the role of specific PKC isozymes in CaP cell growth and survival may aid in development of effective, non-toxic PKC-targeted therapies. ^

Regulation of mucin gene expression by CREB via a nonclassical retinoic acid signaling pathway.

Vitamin A and its metabolite retinoic acid (RA) are essential elements for normal lung development and the differentiation of lung epithelial cells. We previously showed that RA rapidly activated cyclic AMP response element-binding protein (CREB) in a nonclassical manner in normal human tracheobronchial epithelial (NHTBE) cells. In the present study, we further demonstrated that this nonclassical signaling of RA on the activation of CREB plays a critical role in regulating the expression of airway epithelial cell differentiation markers, the MUC2, MUC5AC, and MUC5B genes. We found that RA rapidly activates the protein kinase Calpha isozyme and transmits the activation signal to CREB via the Raf/MEK/extracellular signal-regulated kinase/p90 ribosomal S6 kinase (RSK) pathway. Activated RSK translocated from the cytoplasm to the nucleus, where it phosphorylates CREB. Activated CREB then binds to a cis-acting replication element motif on the promoter (at nucleotides [nt] -878 to -871) of the MUC5AC gene. The depletion of CREB using small interfering RNA abolished not only the RA-induced MUC5AC but also RA-induced MUC2 and MUC5B. Taken together, our findings demonstrate that CREB activation via this nonclassical RA signaling pathway may play an important role in regulating the expression of mucin genes and mediating the early biological effects of RA during normal mucous differentiation in NHTBE cells.

Chromosomal organization and evolutionary conservation of the human chromosome 19q linkage group containing three DNA repair genes

A series of human-rodent somatic cell hybrids were investigated by Southern blot analysis for the presence or absence of twenty-six molecular markers and three isozyme loci from human chromosome 19. Based on the co-retention of these markers in the various independent hybrid clones containing portions of human chromosome 19 and on pulsed field mapping, chromosome 19 is divided into twenty ordered regions. The most likely marker order for the chromosome is: (LDLR, C3)-(cen-MANNB)-D19S7-PEPD-D19S9-GPI-TGF$ \beta$-(CYP2A, NCA, CGM2, BCKAD)-PSG1a-(D19S8, XRCC1)-(D19S19, ATP1A3)-(D19S37, APOC2)-CKMM-ERCC2-ERCC1-(D19S62, D19S51)-D19S6-D19S50-D19S22-(CGB, FTL)-qter.^ The region of 19q between the proximal marker D19S7 and the distal gene coding for the beta subunit of chorionic gonadotropin (CGB) is about 37 Mb in size and covers about 37 cM genetic distance. The ration of genetic to physical distance on 19q is therefore very close to the genomic average OF 1 cM/Mb. Estimates of physical distances for intervals between chromosome 19 markers were calculated using a mapping function which estimates distances based on the number of breaks in hybrid clone panels. The consensus genetic distances between individual markers (established at HBM10) were compared to these estimates of physical distances. The close agreement between the two estimates suggested that spontaneously broken hybrids are as appropriate for this type of study as radiation hybrids.^ All three DNA repair genes located on chromosome 19 were found to have homologues on Chinese hamster chromosome 9, which is hemizygous in CHO cells, providing an explanation for the apparent ease with which mutations at these loci were identified in CHO cells. Homologues of CKMM and TGF$\beta$ (from human chromosome 19q) and a mini-satellite DNA specific to the distal region of human chromosome 19q were also mapped to Chinese hamster 9. Markers from 19p did not map to this hamster chromosome. Thus the q-arm of chromosome 19, at least between the genes PEPD and ERCC1, appears to be a linkage group which is conserved intact between humans and Chinese hamsters. ^

A novel form of CYP3A from rat brain: cDNA cloning, protein characterization and gene regulation of cytochrome P450 3A9

One full length cDNA clone, designated 3aH15, was isolated from a rat brain cDNA library using a fragment of CYP3A2 cDNA as a probe. 3aH15 encoded a protein composed of 503 amino acid residues. The deduced amino acid sequence of 3aH15 was 92% identical to mouse Cyp3a-13 and had a 68.4% to 76.5% homology with the other reported rat CYP3A sequences. Clone 3aH15 was thus named CYP3A9 by Cytochrome P450 Nomenclature Committee. CYP3A9 seems to the major CYP3A isozyme expressed in rat brain. Sexual dimorphism of the expression of CYP3A9 was shown for the first time in rat brain as well as in rat liver. CYP3A9 appears to be female specific in rat liver based on the standards proposed by Kato and Yamazoe who defined sex specific expression of P450s as being a 10-fold or higher expression level in one sex compared with the other. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Male rats treated with estrogen had a similar expression level of CYP3A9 mRNA both in the liver and brain. Ovariectomy of adult female rats drastically reduced the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. On the other hand, only a two-fold induction of CYP3A9 expression by dexamethasone was observed in male liver and no significant induction of CYP3A9 mRNA was observed in female liver or in the brains. These results suggest that estrogen may play an important role in the female specific expression of the CYP3A9 gene and that CYP3A9 gene expression is regulated differently from other CYP3A isozymes. ^ P450 3A9 recombinant protein was expressed in E. coli using the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity (10.1 nmol P450/mg protein) from solubilized fractions using two chromatographic steps. The purified P450 3A9 protein was active towards the metabolism of many clinically important drugs such as imipramine, erythromycin, benzphetamine, ethylmorphine, chlorzoxazone, cyclosporine, rapamycin, etc. in a reconstituted system containing lipid and rat NADPH-P450 reductase. Although P450 3A9 was active towards the catabolism of testosterone, androstenedione, dehydroepiandrosterone (DHEA) and 17β-estradiol, P450 3A9 preferentially catalyzes the metabolism of progesterone to form four different hydroxylated products. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture and GSH. The possible mechanisms of the stimulatory effects of GSH on P450 3A9 activities are discussed. Sexually dimorphic expression of P450 3A9 in the brain and its involvement in many neuroactive drugs as well as neurosteroids suggest the possible role of P450 3A9 in some mental disorders and brain functions. ^

CYTOCHROME P450 MEDIATED REDUCTIVE DEHALOGENATION OF HEXACHLOROBENZENE

The role of the cytochrome (CYT) P-450 mixed-function oxidase (MFO) in the biotransformation of hexachlorobenzene (HCB) was investigated, since in vivo interaction between this enzyme and chemical is very probable. HCB is a type I substrate with (Fe('3+)) CYT P-450 isozymes present in untreated, b-naphthoflavone (BNF) and phenobarbital (PB) induced rat liver microsomes. HCB dependent and saturable type I binding titrations yield spectral dissociation constants (K(,s)) of 180 and 83 uM for the isozymes present in untreated and PB induced microsomes, respectively. Purified CYT P-450b, the major isozyme induced by PB, produces HCB dependent and saturable type I spectra with a K(,s) of 0.38 uM.^ CYT P-450 mediated reductive dehalogenation occurs in microsomes and purified/reconstituted MFO systems and produces pentachlorobenzene (PCB) as the initial and major metabolite under both aerobic and anaerobic conditions. In microsomal reactions secondary metabolism of PCB occurs in the presence of oxygen. Pentachlorophenol (PCP) is produced only in aerobic reactions with PB induced microsomes with a concomitant decrease in PCB production. PCP is not detected in aerobic reactions with BNF induced microsomes, although PCB production is decreased compared to anaerobic conditions. A reaction scheme for the production of phenolic metabolities from PCB is deduced.^ CYT P-450 dependent and NADPH independent modes of PCB production occur with purified/reconstituted MFO systems and are consistent with dehalogenation pathways observed with microsomal experiments. The NADPH independent production of PCB requires native microsomal or purified MFO protein components and may be the result of nucleophilic displacement of a chlorine atom from HCB mediated or coupled with redox active functions (primary, secondary, tertiary and quarternary structures) of the proteins. CYT P-450 dependent production of PCB from HCB is isozyme dependent: CYT P-450c = CYT P-450d > CYT P-450a > CYT 450b. The low apparent specific activity may be due to non-optimal reconstitution conditions (e.g., isozyme choice and requirement of other microsomal elecron transport components) and secondary metabolism of PCB and the phenols derived from PCB. CYT P-450 mediated dehalogenation may be catalyzed through attack, by the iron oxene (postulated intermediate of CYT P-450 monooxygenations), at the chlorines of HCB instead of the aromatic nucleus. (Abstract shortened with permission of author.) ^

THE MICROCELL-MEDIATED TRANSFER OF SINGLE HUMAN CHROMOSOMES INTO RECIPIENT MOUSE CELLS

Microcell-mediated chromosome transfer is a method of gene transfer which allows for the introduction of single or small groups of intact chromosomes into recipient host cells. Microcell transfer was first performed by Fournier and Ruddle using rodent microcells and various recipient cells. Expansion of this technology to include the transfer of normal human genetic material has been hindered because large micronucleate populations from diploid human cells have been unobtainable. This dissertation research describes, however, the methods for production of micronuclei in 40-60% of normal human fibroblasts. Once micronucleate cells were obtained, they were enucleated by centrifugation in the presence of Cytochalasin B; the microcells were then purified and fused to recipient mouse (LMTK('-)) cells using a new fusion protocol employing polyethylene glycol containing phytohemagglutinin. Microcell clones were isolated from the HAT selection system. Alkaline Giemsa staining performed on these hybrids indicated the presence of a single human chromosome in each of seven microcell clones from three separate experiments. That chromosome was further identified by G banding analysis to be human chromosome #17, which codes for thymidine kinase. The time course for production of these hybrids from fusion to karyotypic analysis was 6 weeks. The viability of the transferred human genetic material was assessed by electrophoretic isozyme analysis.^ Subsequent experiments were performed in an attempt to optimize the transfer frequency for the thymidine kinase gene using this system. Results indicated that the frequency could be increased from < 1 x 10('-6) in initial experiments to 2 x 10('-5) in the latest experiment. Analyses were also conducted to determine the number of chromosomes per isolated microcell as well as to investigate the stability of the transferred human chromosome in the mouse genome. ^

ARRANGEMENT OF GENE LOCI IN EUPLOID CHINESE HAMSTER CELLS AND GENETIC CONSEQUENCES OF REARRANGEMENT IN CHO CELLS

In both euploid Chinese hamster (Cricetulus griseus) cells and pseudodiploid Chinese hamster ovary (CHO) cells, gene assignments were accomplished by G band chromosome and isozyme analysis (32 isozymes) of interspecific somatic cell hybrids obtained after HAT selection of mouse CL 1D (TK('-)) cells which were PEG-fused with either euploid Chinese hamster cells or HPRT('-) CHO cells. Hybrids slowly segregated hamster chromosomes. Clone panels consisting of independent hybrid clones and subclones containing different combinations of Chinese hamster chromosomes and isozymes were established from each type of fusion.^ These clone panels enabled us to provisionally assign the loci for: nucleoside phosphorylase (NP), glyoxalase (GLO), glutathione reductase (GSR), adenosine kinase (ADK), esterase D (ESD), peptidases B and S (PEPB and -S) and phosphoglucomutase 2 (PGM2, human nomenclature) to chromosome 1; adenylate kinase 1 (AK1), adenosine deaminase (ADA) and inosine triosephosphatase (ITP) to chromosome 6; triosephosphate isomerase (TPI) to chromosome 8; and glucose phosphate isomerse (GPI) and peptidase D (PEPD) to chromosome 9.^ We also confirm the assignments of 6-phosphogluconate dehydrogenase (PGD), PGM1, enolase 1 (ENO1) and diptheria toxin sensitivity (DTS) to chromosome 2 as well as provisionally assign galactose-1-phosphate uridyl transferase (GALT) and AK2 to chromosome 2. Selection in either HAT or BrdU for hybrids that had retained or lost the chromosome carrying the locus for TK enabled us to assign the loci for TK, galactokinase (GALK) and acid phosphatase 1 (ACP1) to Chinese hamster chromosome 7.^ These results are discussed in relation to current theories on the basis for high frequency of drug resistant autosomal recessive mutants in CHO cells and conservation of mammalian autosomal linkage groups. ^

The regulation of protein kinase C by thiol oxidation

The Ser/Thr protein kinase C (PKC) isozyme family plays an important role in cell growth and differentiation and also contributes to key events in the development and progression of cancer. PKC isozymes are activated by phospholipid-dependent mechanisms, and they are also subject to oxidative activation and inactivation. Oxidative regulatory mechanisms are important in the governance of PKC isozyme action. While oxidative PKC activation involves phospho-tyrosine (P-Y) stabilization, the molecular mechanism(s) for oxidative PKC inactivation have not been defined. We previously reported that Thr → Cys peptide-substrate analogs inactivate several PKC isozymes including PKC-α via S-thiolation, i.e., by forming disulfides with PKC thiols. This inactivation mechanism is chemically analogous to protein S-glutathiolation, a post-translational modification that has been shown to oxidatively regulate several enzymes. To determine if PKC-α could be inactivated by S-glutathiolation, we employed the thiol-specific oxidant diamide (0.01–10mM) and 100μM glutathione (GSH). Diamide alone (0.1–5.0 mM) weakly inactivated PKC-α (<20%), and GSH alone had no effect on the isozyme activity. Marked potentiation of diamide-induced PKC-α inactivation (>90%) was achieved by 100μM GSH, resulting in full inactivation of the isozyme. Inactivation was reversed by DTT, consistent with a mechanism involving PKC-α S-glutathiolation. S-glutathiolation was demonstrated as DTT-reversible incorporation of [35S] GSH into PKC-α isozyme structure. These results indicate that a mild oxidative stimulus can inactivate purified PKC-α via S-glutathiolation. In addition, diamide treatment of metabolically labeled NIH3T3 cells induced potent PKC-α inactivation via isozyme [35S] S-thiolation. These results indicate that cellular PKC-α can be regulated via S-glutathiolation. ^

Inactivation and self -association of CaM kinase: Effects of ATP, substrate binding domains, and isozymic forms

Calcium/calmodulin-dependent protein kinase II (CaM kinase) is a multifunctional Ser/Thr protein kinase, that is highly enriched in brain and is involved in regulating many aspects of neuronal function. We observed that forebrain CaM kinase from crude homogenates, cytosolic fractions and purified preparations inactivates and translocates into the particulate fraction following autophosphorylation. Using purified forebrain CaM kinase as well as recombinant $\alpha$ isozyme, we determined that the formation of particulate enzyme was due to enzyme self-association. The conditions of autophosphorylation determine whether enzyme self-association and/or inactivation will occur. Self-association of CaM kinase is sensitive to pH, ATP concentration, and enzyme autophosphorylation. This process is prevented by saturating concentrations of ATP. However, in limiting ATP, pH is the dominant factor, and enzyme self-association occurs at pH values $\rm{<}7.0.$ Site-specific mutants were produced by substituting Ala for Thr286, Thr253, or Thr305,306 to determine whether these sites of autophosphorylation affect enzyme inactivation and self-association. The only mutation that influenced these processes was Ala286, which removed the protective effect afforded by autophosphorylation in saturating ATP. Enzyme inactivation occurs in the presence and absence of self-association and appears predominantly sensitive to nucleotide concentration, because saturating concentrations of $\rm Mg\sp{2+}/ADP$ or $\rm Mg\sp{2+}/ATP$ prevent this process. These data implicate the ATP binding pocket in both inactivation and self-association. We also observed that select peptide substrates and peptide inhibitors modeled after the autoregulatory domain of CaM kinase prevented these processes. The $\alpha$ and $\beta$ isozymes of CaM kinase were characterized independently, and were observed to exhibit differences in both enzyme inactivation and self-association. The $\beta$ isozyme was less sensitive to inactivation, and was never observed to self-associate. Biophysical characterization, and transmission electron microscopy coupled with image analysis indicated both isozymes were multimeric, however, the $\alpha$ and $\beta$ isozymes appeared structurally different. We hypothesize that the $\alpha$ subunit of CaM kinase plays both a structural and enzymatic role, and the $\beta$ subunit plays an enzymatic role. The ramifications for the functional differences observed for inactivation and self-association are discussed based on potential structural differences and autoregulation of the $\alpha$ and $\beta$ isozymes in both calcium-induced physiological and pathological processes. ^