Expression and activation of protein kinase C isoforms in a human megakaryocytic cell line

Megakaryocytes undergo a unique differentiation program, becoming polyploid through repeated cycles of DNA synthesis without concomitant cell division. We have shown previously that phorbol 12-myristate 13-acetate (PMA) induces the Dami human megakaryocytic cell line to become polyploid and to express platelet-specific proteins, including von Willebrand factor (vWF) and glycoprotein Ib (GpIb). Phorbol esters are thought to regulate gene expression principally through the activation of protein kinase C (PKC), a family of structurally related kinases with potentially unique activation requirements and substrate specificities. A survey of PKC isoforms in Dami cells revealed that, by both Western and Northern analyses, PKC isoforms alpha, beta, delta, epsilon, eta, theta, and zeta were reproducibly detected. PKC-gamma was not detected. In order to define the role of individual PKC isoforms in megakaryocytic maturation, PMA and 2-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a putative selective activator of the PKC-beta 1 isotype, were compared for their effects on Dami cell maturation. Treatment with either dPPA or PMA caused Dami cells to cease proliferating, to become polyploid, and to express vWF. We also examined dPPA and PMA for their ability to activate and to downregulate expression of different PKC isoforms. Fifteen-minute treatment with PMA resulted in the translocation of PKC isoforms alpha, epsilon, and theta from the cytosolic to the membrane fraction; twenty-four hour treatment resulted in the downregulation of these isoforms. In contrast, dPPA was found to be a potent activator of PKC-epsilon alone and exhibited weaker effects on alpha and theta. These data suggest that PKC isoforms beta, delta, eta, and zeta, which appear not to be activated by either phorbol ester, are unlikely to be primarily involved in megakaryocytic maturation in response to these agents. The isoforms that are translocated by both phorbol esters-PKC isoforms alpha and theta, and particularly epsilon-are more likely to transduce the signals that stimulate Dami cell differentiation.     

Modulation of MAP kinase signaling and growth characteristics by the overexpression of protein kinase C in NIH3T3 cells

This study was performed to examine effects of the overexpression of protein kinase C (PKC) isoforms (i.e., beta I, beta II, gamma, delta, eta, and zeta) on mitogen-activated protein (MAP) kinase (Erk-1 and -2) signaling and growth characteristics of NIH3T3 cells. Phorbol ester (PMA) activated endogenous and ectopically expressed PKC alpha, beta I, beta II, gamma, delta, epsilon, and eta. Overexpression of the examined PKC isoforms enhanced PMA-induced MAP kinase activation. Potentiation of MAP kinase activation was also observed upon stimulation of cells with platelet-derived growth factor (PDGF) although there was no indication for the activation PKC isoforms by PDGF. Inhibition of PKC blocked PMA- but not PDGF-induced MAP kinase activation. Thus, potentiation of PDGF-induced MAP kinase activation appears to be independent to PKC activity, while PMA-induced MAP kinase activation requires PKC activity. The ability of PKC isoforms to potentiate MAP kinase activation is not related to the growth characteristics of cells because individual PKC isoforms differentially regulated maximum density and proliferation of cells.     

Erythropoietin stimulates nuclear localization of diacylglycerol and protein kinase C beta II in B6SUt.EP cells

Erythropoietin (EPO) is a hormone, as well as a hematopoietic growth factor, that specifically regulates the proliferation and differentiation of erythroid progenitor cells. Although the membrane-bound receptor for EPO has no intrinsic kinase activity, it triggers the activation of protein kinases via phospholipases A2, C, and D. A cascade of serine and threonine kinases, including Raf-1, MAP kinase and protein kinase C (PKC) is activated following tyrosine phosphorylation. In this study, we have examined whether changes in nuclear PKC and 1,2-diacylglycerol (DAG) are induced following EPO treatment of the murine target cell line, B6SUt.EP. Western blot analysis using isoform-specific antibodies demonstrated the presence of PKC beta II, but not PKC alpha, beta I, gamma, epsilon, delta, eta, or zeta in the nuclei of cells stimulated with EPO. The increase in nuclear beta II levels was accompanied by an immediate rise in DAG mass levels with both of the increases peaking by 1 min. These rapid increases in nuclear DAG and PKC beta II expression suggest a mechanism for EPO-induced changes in gene expression necessary for cell proliferation.     

Selective regulation of protein kinase C isoenzymes by oleic acid in human platelets

Cis-unsaturated fatty acids activate soluble protein kinase C (PKC) in vitro and in intact platelets. The following studies were conducted to determine the effects of oleate on individual isoenzymes of PKC in human platelets. Human platelets were found to contain predominantly PKC alpha, beta I, beta II, and delta with minor immunoreactivity for PKC epsilon, zeta, and eta. In intact platelets, sodium oleate caused a time-dependent redistribution of PKC alpha, beta II, and delta from cytosol to membrane fractions with little effects on PKC beta I. On the other hand, PMA and thrombin induced translocation of all four isoenzymes of PKC. In vitro, oleate partially activated (50% of Vmax) purified calcium-dependent PKC (alpha, beta I, and beta II) with an EC50 of 50 microM whereas it fully activated (100% of Vmax) purified calcium-independent PKC (predominantly delta) with an EC50 of 5 microM. The selective effects of oleate on PKC isoenzymes were investigated in platelet cytosol which contains endogenous PKC and its physiologic substrates. Under these conditions, oleate potently activated calcium-independent PKC causing the phosphorylation of the 40-kDa substrate. Activation of calcium-dependent isoforms occurred only at higher concentrations of oleate. Thus, oleate activates multiple isoenzymes of PKC with predominant effects on calcium-independent PKC.     

Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells

The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.     

Salivary cortisol and cardiovascular activity during stress in oppositional-defiant disorder boys and normal controls

The erythroleukemic cell line K562 can undergo further differentiation in erythroid or megakaryocytic lineage depending on the nature of the stimulus. Phorbol ester (PMA) stimulates megakaryocytic development whereas hemin promotes erythroid differentiation of these cells. We have examined the effect of PMA and hemin on the expression of the Kell blood group and CD10 antigens, two related proteins that belong to a family of membrane-bound neutral metalloendopeptidases. We show here that differentiation of K562 cells by PMA in the megakaryocytic lineage results in abolishment of Kell mRNA accumulation and protein expression and, in parallel, the induction of CD10 mRNA accumulation, protein expression, and enzymatic activity. Conversely, differentiation of these cells by hemin in the erythroid lineage is accompanied by an up-regulation of Kell mRNA and protein expression, with no changes in CD10 mRNA and protein expression. Thus, CD10 and Kell can be regarded as specific markers of the differentiation of K562 cells in the megakaryocytic and erythroid lineages, respectively.     

Inhibitory effects of an antiestrogen, toremifene, on the phorbol ester-induced adhesive capacity of breast carcinoma cells

Previous studies have revealed that protein kinase C (PKC) is responsible for malignant progression. In the present study, we investigated the potent inhibitory effects of an antiestrogen, toremifene, on PKC-mediated cellular adhesion. A phorbol ester, phorbol 12-myristate 13-acetate (PMA), significantly enhanced alpha2beta1 integrin-dependent adhesion of MCF-7 breast carcinoma cells. This PMA-induced adhesion was partially inhibited by incubating cells with toremifene prior to PMA exposure in a time- and dose-dependent manner. FACS analysis demonstrated that the PMA-induced alpha2beta1-dependent cellular adhesion was accompanied with elevated expression of alpha2beta1+integrin subunit on the cell surface. However, toremifene did not affect the elevated expression levels of these integrins but rather the avidity of alpha2beta1 integrin. We concluded that toremifene inhibited cellular adhesion activated by PMA, probably through mechanism which inhibits PKC.     

Fibronectin-mediated cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation. The signaling role of protein kinase C-beta

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-beta-deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor alpha5 beta1 integrin. HL-525 cells, which constitutively display high levels of surface alpha5 beta1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that alpha5 beta1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.     

Protein kinase C isozymes in human megakaryoblastic leukemia cell line, MEG-01: possible involvement of the isozymes in the differentiation process of MEG-01 cells

The expression of the different protein kinase C (PKC) isozymes in various states of differentiation of the human megakaryoblastic leukaemia cell line MEG-01 were analysed using thermocycle amplification of mRNA and immunoblotting. MEG-01 expressed mRNAs of PKC alpha, -beta I, -beta II, -delta, -epsilon, -eta, -theta and -zeta, but not PKC gamma. At the protein molecule level, MEG-01 was observed to express PKC alpha, -beta I, -beta II,- epsilon, -theta and -zeta, but lack -gamma, -delta and -eta. When differentiation of MEG-01 was induced by 100 nm 12-O-tetradecanoyl-phorbol-13-acetate (TPA), rapid translocation from cytosol to membrane fraction and down-regulation of PKC alpha, -epsilon and -theta was observed in 1-2h. On the other hand, PKC beta I and -beta II were observed to translocate not only to the membrane fraction but also to the cytoskeletal fraction in a different manner, and their down-regulation, especially beta II, was very slow. The myristoylated, alanine-rich C kinase substrate (MARCKS) in the membrane fraction of MEG-01 cells was observed to decrease gradually throughout the differentiation process. Additionally, time-course study of TPA treatment indicated that incubation of the cells for 30 min is sufficient for differentiation. These results strongly suggest that the activation of PKC alpha, -epsilon and -theta is involved in the initiation of differentiation, and that PKC beta I and -beta II have important roles in the maintenance of differentiation. Although PKC zeta was resistant to TPA treatment and its translocation was reduced, the amount of this isozyme in the cytosol fraction decreased throughout the differentiation process.     

Protein kinase C is not necessary for transforming growth factor beta-induced growth-arrest in leukemia cell lines

Growth and differentiation of blood cell precursors are regulated by cytokines and hormones by mechanisms which are incompletely understood. Protein kinase C (PKC) isozymes are widely regarded as being important in signal transduction pathways. We have shown that one isozyme, PKC beta, is uniquely important in mediating phorbol ester-induced growth-arrest in the HL-60 myeloid cell line. 1,25-dihydroxyvitamin D3 induces differentiation and growth-arrest in many cells. It upregulates the expression of PKC beta, potentiating the action of phorbol ester. We tested the hypotheses that cytokines, which arrest the growth of hematopoietic cells, do so by activating PKC beta, and that differentiation and growth-arrest induced by 1,25-dihydroxyvitamin D3 is caused by upregulation of PKC beta isozyme gene expression. The influence on growth of combinations of five cytokines (TNF alpha, TGF beta 1, gamma-IFN, IL-1, and G-CSF) and 1,25-dihydroxyvitamin D3 on ten human leukemia cell lines (THP-1, HL-60 S, HL-60 PET, U937, K562, Jurkat, MOLT-4, RPM1 8402, KG-1, and KG-1a) was determined. Four cell lines (THP-1, HL-60 S and PET, and U937) exhibited total growth-arrest when incubated with 1,25-dihydroxyvitamin D3 followed by TGF beta 1. The expression by each cell line of mRNA encoding PKC alpha, beta, and delta, both before and after 24 or 48 h of incubation with 1,25-dihydroxyvitamin D3, was determined. Cell lines sensitive to TGF beta 1 each expressed PKC delta endogenously, or expression was up-regulated with 1,25-dihydroxyvitamin D3. U937 cells underexpressed PKC alpha, and HL-60 PET cells underexpressed PKC beta. These data suggested that PKC delta could be responsible for mediating growth-arrest by TGF beta 1. To test this hypothesis directly, we incubated the cells with two bisindolylmaleimide PKC inhibitors during the addition of 1,25-dihydroxyvitamin D3 and TGF beta 1. Surprisingly, the PKC inhibitors did not block the growth-arrest induced by 1,25-dihydroxyvitamin D3 and TGF beta 1. This experiment strongly suggests that neither growth-arrest induced by TGF beta 1 nor the potentiation of this growth-arrest by 1,25-dihydroxyvitamin D3 is mediated by a PKC isozyme which is inhibitable by the bisindolymaleimides.     

Linkage of protein kinase C-beta activation and intracellular interleukin-2 accumulation in human naive CD4 T cells

A critical role for protein kinase C (PKC) in signal transduction events has been well established. Moreover, studies of regulation in PKC levels suggest participation in mediating long-term cellular functions. Protein kinase C-beta (PKC-beta) has been reported to be involved in interleukin-2 (IL-2) synthesis in T lymphocytes. In this study, the role of PKC-beta in intracellular accumulation of IL-2 was investigated using specific inhibitors. Preincubation with two different PKC inhibitors, one specific for classical isotypes (alpha and beta I) Go6976, and one which inhibits both classical and non-classical isotypes, GF109203X, caused a complete block in cytoplasmic IL-2 accumulation when naive CD4 T cells were stimulated in the presence of CD2+CD28+phorbol myristate acetate (PMA). In contrast, preincubation with up to 1000 ng/ml of cyclosporin A (CsA) resulted in a reduction in the intracellular IL-2 detected, as observed by a decrease in the proportion of positive cells as well as a fall in the mean fluorescence intensity (MFI). CsA did not influence PKC-beta translocation. Flow cytometric assessments of PKC-beta and its isoforms beta I and beta II correlated with Western blotting analysis and these results were further supported by the use of PKC-beta-positive (HUT 78) and -negative (BW5147) T-cell lines. Using the specific inhibitors, Go6976 and GF109203X, the findings in this study suggest that activation and translocation of PKC-beta is critical for accumulation of intracellular IL-2. The influence of CsA in reducing but not blocking IL-2 synthesis is discussed. PMA-induced down-regulation of the CD4 antigen was observed in the presence of Go6976 and but not GF109203X, suggesting regulation by non-classical PKC isoforms.     

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.     

Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C

Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the alpha, delta, eta, and zeta isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the alpha, delta, and eta isoforms. Overexpression of the eta isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The eta-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the eta-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the eta isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative eta isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1alpha,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the delta isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the alpha and zeta isoforms did not. These findings indicate that the eta and delta isoforms of PKC are involved crucially in squamous cell differentiation.     

The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase C-zeta

The heparin-binding protein vascular endothelial growth factor (VEGF) is a highly specific growth factor for endothelial cells. VEGF binds to specific tyrosine kinase receptors, which mediate intracellular signaling. We investigated 2 hypotheses: (1) VEGF affects intracellular calcium [Ca2+]i regulation and [Ca2+]i-dependent messenger systems; and (2) these mechanisms are important for VEGF's proliferative effects. [Ca2+]i was measured in human umbilical vein endothelial cells using fura-2 and fluo-3. Protein kinase C (PKC) activity was measured by histone-like pseudosubstrate phosphorylation. PKC isoform distribution was observed with confocal microscopy and Western blot. Inhibition of PKC isoforms was assessed by specific antisense oligonucleotides (ODN) for the PKC isoforms. VEGF (10 ng/mL) induced a transient increase in [Ca2+]i followed by a sustained elevation. The sustained [Ca2+]i plateau was abolished by EGTA. Pertussis toxin also abolished the plateau phase, whereas the initial peak was not affected. The PKC isoforms alpha, delta, epsilon, and zeta were identified in endothelial cells. VEGF induced a translocation of PKC-alpha and PKC-zeta toward the nucleus and the perinuclear area, whereas cellular distribution of PKC-delta and PKC-epsilon was not influenced. Cell exposure to TPA led to a down-regulation of PKC-alpha and reduced the proliferative effect of VEGF. VEGF-induced endothelial cell proliferation also was reduced by the PKC inhibitors staurosporine and calphostin C. Specific down-regulation of PKC-alpha and PKC-zeta with antisense ODN reduced the proliferative effect of VEGF significantly. Our data show that VEGF induces initial and sustained Ca2+ influx. VEGF leads to the translocation of the [Ca2+]i-sensitive PKC isoform alpha and the atypical PKC isoform zeta. Antisense ODN for these PKC isoforms block VEGF-induced proliferation. These findings suggest that PKC isoforms alpha and zeta are important for VEGF's angiogenic effects.     

Bovine protein kinase C isotypes transduced into Molt-4/HIV-1 cells augment the expression of HIV-1 genome

Protein kinase C (PKC) is known to be an ubiquitous enzyme found in a variety of mammalian tissue and has been shown to require Ca2+ and phospholipid, and to be further activated by diacylglycerol, which increased the affinity of the kinase for both Ca2+ and phospholipid. PKC is composed with a family of multiple isotypes with closely related structures, although the detailed functions of each isotype have not yet been clarified. We tried to clarify the difference of biological functions among the isotypes of PKC. Three PKC isotypes (alpha, beta 1, gamma) were purified from bovine brain by L-threonine-Sepharose affinity column, and fractions containing alpha and beta 1 forms (Peak A) and gamma form (Peak B) were obtained. Each peak integrated into erythrocytic ghost was transduced into cells persistently infected with HIV-1 (Molt-4/HIV-1) by fusion method. Peak A containing PKC alpha and beta 1 isotypes induced TNF-alpha production, HIV-1 cDNA titer and P24 antigen in culture of Molt-4/HIV-1 cells, but Peak B containing PKC gamma did not show any effects on Molt-4/HIV-1 cells. The effect of PKC alpha and beta 1 on the production of TNF-alpha and the augmentation of HIV-1 replication are inhibited by both anti-TNF-alpha antibody and staurosporine, a potent PKC inhibitor.     

The zeta isoform of protein kinase C controls interleukin-2-mediated proliferation in a murine T cell line: evidence for an additional role of protein kinase C epsilon and beta

In order to address a role of protein kinase C in signal transduction through interleukin-2, interleukin-4, and interleukin-9 receptors, we took advantage of the availability of a selective protein kinase C inhibitor, GF109203X, and the availability of TS1 beta and TS1 alpha beta cell lines which can be maintained in interleukin-2, interleukin-4, or interleukin-9 independently. In this report we report that inhibition of protein kinase C activity by GF109203X does not block interleukin-4- or interleukin-9-dependent proliferation and, on the contrary, does block interleukin-2-dependent proliferation, suggesting that interleukin-4 and interleukin-9 do not use signal transduction pathways mediated by protein kinase C and that the common gamma chain of interleukin-2, interleukin-4, and interleukin-9 receptors is not responsible per se for the activation of protein kinase C through interleukin-2 receptor. Moreover, GF109203X induces apoptosis in cells cultured in interleukin-2 but not in interleukin-4 or interleukin-9. Using antisense oligonucleotides, we report that the zeta and epsilon protein kinase C isoforms are involved in signaling through high-affinity interleukin-2 receptor and beta and zeta are involved in signaling through intermediate-affinity interleukin-2 receptor. Taken together, our data indicate that activation of the zeta, beta, and epsilon protein kinase C isoforms is an important step in interleukin-2-mediated proliferation.