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S H Yuspa

Publications and source records attributed to S H Yuspa.

At least 19 recordsLinked to original sources
PubMed2001

Protein kinase Cdelta-mediated phosphorylation of alpha6beta4 is associated with reduced integrin localization to the hemidesmosome and decreased keratinocyte attachment.

In mammalian epidermis, expression of the alpha6beta4 integrin is restricted to the hemidesmosome complexes, which connect the proliferative basal cell layer with the underlying basement membrane. Keratinocyte differentiation is associated with down-regulation of alpha6beta4 expression and detachment of keratinocytes from the basement membrane. Neoplastic keratinocytes delay maturation, proliferate suprabasally, and retain the expression of the alpha6beta4 integrin in suprabasal cells disassociated from the hemidesmosomes. We now show that the alpha6beta4 integrin is a substrate for serine phosphorylation by protein kinase C in keratinocytes. Furthermore, protein kinase C-mediated phosphorylation of alpha6beta4 is associated with redistribution of this integrin from the hemidesmosome to the cytosol. Specifically, in vitro kinase assays identified the protein kinase Cdelta as the primary isoform phosphorylating alpha6 and beta4 integrin subunits. Using recombinant protein kinase C adenoviruses, overexpression of protein kinase Cdelta but not protein kinase Calpha in primary keratinocytes increased beta4 serine phosphorylation, decreased alpha6beta4 localization to the hemidesmosome complexes, and reduced keratinocyte attachment. Taken together, these results establish a link between protein kinase Cdelta-mediated serine phosphorylation of alpha6beta4 integrin and its effects on alpha6beta4 subcellular localization and keratinocyte attachment to the laminin underlying matrix.

Animals↗
PubMed2001

Overexpression of retinoic acid receptors alpha and gamma into neoplastic epidermal cells causes retinoic acid-induced growth arrest and apoptosis.

Retinoids are essential for normal epidermal differentiation and are used for the prevention and treatment of numerous skin disorders and cancers in humans. In previous studies, we have shown that retinoic acid receptors (RARs) -alpha and -gamma are down-regulated during skin tumor progression. The transduction of v-ras(Ha) into primary mouse keratinocytes is sufficient to reduce both RARalpha and RARgamma protein levels as well as inhibit their transactivation functions. Our primary objective is to investigate the roles that RARalpha and RARgamma play in keratinocyte tumor cell proliferation. Through retroviral gene transduction, we overexpressed RARalpha or RARgamma into neoplastic mouse epidermal cells with down-regulated endogenous RAR proteins. Following all-trans retinoic acid (RA) treatment, RARalpha- and RARgamma-transduced cell lines exhibit a progressive, dose-dependent growth inhibition relative to the control LXSN cell lines. Further characterization of RAR-transduced cells following RA treatment reveals that both RARalpha and RARgamma cause a decrease in S-phase population, while only RARalpha causes a simultaneous G(0)/G(1) block as evidenced by reduced [(3)H]-thymidine incorporation and flow cytometric analysis of DNA content. Following RA treatment, both receptors cause an early, transient increase in the cyclin-dependent kinase inhibitor (CDKI) p21 proteins, while only RARalpha causes a simultaneous sharp, brief increase in the CDKI p16 protein. A later decrease in cyclin D(1) protein is also evident in RARalpha- and RARgamma-transduced cells. Chromatin condensation and PARP cleavage are observed in both RARalpha- and RARgamma-transduced cells indicating an RA-induced apoptosis that may be caspase dependent. Furthermore, both receptors cause a late upregulation and apparent cleavage of the squamous differentiation marker protein kinase C (PKC)-eta. These results suggest that RARalpha and RARgamma enhance growth suppression and apoptosis of neoplastic epidermal keratinocytes. This growth inhibitory effect of both retinoid receptors in neoplastic keratinocytes may be achieved through distinct as well as overlapping mechanisms of cell cycle control.

Animals↗
PubMed2000

Activator protein 1 transcription factors are fundamental to v-rasHa-induced changes in gene expression in neoplastic keratinocytes.

The induction of mouse skin papillomas by initiation-promotion protocols is associated with aberrant expression of epithelial markers in the tumor mass. Similarly, initiation of mouse keratinocytes with a retrovirus encoding the v-rasHa gene (v-rasHa keratinocytes) causes characteristic alterations of epidermal gene expression (A. A. Dlugosz et at, Cancer Res., 54: 6413-6420, 1994). Because activator protein 1 (AP-1) proteins are likely targets of Ras activation, we have examined the role of AP-1 factors in v-rasHa keratinocytes. Introduction of v-rasHa into keratinocytes up-regulates c-Fos, deltaFos B, and Fra-1 transcripts and protein levels in nuclear extracts. The expression of Jun proteins is not significantly altered in v-rasHa keratinocytes. Transduction of cells with v-rasHa results in increased AP-1-dependent transcriptional activity, which is also simulated by transfection of keratinocytes with either c-Fos or deltaFos B but not Fra-1, suggesting that the up-regulation of c-Fos and deltaFos B contributes to this effect. To explore the role of AP-1 proteins in regulating keratinocyte markers in v-rasHa keratinocytes, we blocked the binding of AP-1 proteins to DNA by infecting keratinocytes with an adenovirus encoding a dominant-negative Fos mutant (A-FOS). A-FOS replaces endogenous Fos proteins in the formation of heterodimers with Jun family members and thus prevents the AP-1 transcription factor from binding to DNA. In v-rasHa keratinocytes, the A-FOS virus reversed the suppression of keratins 1 and 10 transcripts and protein, which is characteristically seen in tumors and v-rasHa keratinocytes. A-FOS also increased protein levels but reduced transcripts for the late marker, loricrin, a component of the cornified envelope. These findings indicate that AP-1 proteins are involved in the changes in gene expression that define the v-rasHa phenotype in mouse keratinocytes.

Animals↗
PubMed2000

The epidermal growth factor receptor is required to maintain the proliferative population in the basal compartment of epidermal tumors.

Previous studies using keratinocytes from epidermal growth factor receptor (EGFR)-deficient mice revealed that the EGFR is not required for papilloma formation initiated by a mutant rasHa gene, although the tumors that develop are very small (A. A. Dlugosz et aL, Cancer Res., 57: 3180-3188, 1997). The current study used a combination of bromodeoxyuridine pulse-chase, proliferating cell nuclear antigen distribution, and differentiation marker analysis to reveal the following: (a) the EGFR was required to maintain the proliferative population in the basal cell compartment of papillomas; (b) in the absence of EGFR, cycling tumor cells migrated into the suprabasal compartment and initiated the differentiation program prematurely; and (c) these changes were associated with cell cycle arrest. Further analysis of v-rasHa-transformed EGFR-deficient keratinocytes in vitro indicated that such cells migrated more on and attached less to extracellular matrix components. Together, these studies reveal that an essential function for the EGFR pathway in squamous tumors is to maintain a proliferative pool of basal cells and prevent premature terminal differentiation.

Animals↗
PubMed2000

Involvement of protein kinase C delta (PKCdelta) in phorbol ester-induced apoptosis in LNCaP prostate cancer cells. Lack of proteolytic cleavage of PKCdelta.

Phorbol esters, the activators of protein kinase C (PKC), induce apoptosis in androgen-sensitive LNCaP prostate cancer cells. The role of individual PKC isozymes as mediators of this effect has not been thoroughly examined to date. To study the involvement of the novel isozyme PKCdelta, we used a replication-deficient adenovirus (PKCdeltaAdV), which allowed for a tightly controlled expression of PKCdelta in LNCaP cells. A significant reduction in cell number was observed after infection of LNCaP cells with PKCdeltaAdV. Overexpression of PKCdelta markedly enhanced the apoptotic effect of phorbol 12-myristate 13-acetate in LNCaP cells. PKCdelta-mediated apoptosis was substantially reduced by the pan-caspase inhibitor z-VAD and by Bcl-2 overexpression. Importantly, and contrary to other cell types, PKCdelta-mediated apoptosis does not involve its proteolytic cleavage by caspase-3, suggesting that allosteric activation of PKCdelta is sufficient to trigger apoptosis in LNCaP cells. In addition, phorbol ester-induced apoptosis was blocked by a kinase-deficient mutant of PKCdelta, supporting the concept that PKCdelta plays an important role in the regulation of apoptotic cell death in LNCaP prostate cancer cells.

Apoptosis↗
PubMed2000

Cross-talk between epidermal growth factor receptor and protein kinase C during calcium-induced differentiation of keratinocytes.

The induction of epidermal differentiation by extracellular Ca2+ involves activation of both tyrosine kinase and protein kinase C (PKC) signaling cascades. To determine if the differentiation-dependent activation of tyrosine kinase signaling can influence the PKC pathway, we examined the tyrosine phosphorylation status of PKC isoforms in primary mouse keratinocytes stimulated to terminally differentiate with Ca2+. Elevation of extracellular Ca2+ induced tyrosine phosphorylation of PKC-delta, but not the other keratinocyte PKC isoforms (alpha, epsilon, eta, zeta). We have previously demonstrated that activation of the epidermal growth factor receptor (EGFR) pathway induces PKC-delta tyrosine phosphorylation in basal keratinocytes (Denning M F, Dlugosz A A, Threadgill D W, Magnuson T, Yuspa S H (1996) J Biol Chem 271: 5325-5331). When basal keratinocytes were stimulated to differentiate by Ca2+, the level of cell-associated transforming growth factor-alpha (TGF-alpha) increased 30-fold, while no increase in secreted TGF-alpha was detected. Furthermore, Ca2+-induced tyrosine phosphorylation of PKC-delta and phosphotyrosine-association of the receptor adapter protein Shc was diminished in EGFR -/- keratinocytes, suggesting that EGFR activation may occur during keratinocyte differentiation. Tyrosine phosphorylated PKC-delta was also detected in mouse epidermis, suggesting that this differentiation-associated signaling pathway is physiological. These results establish a requirement for the EGFR in Ca2+-induced tyrosine phosphorylation of PKC-delta, and document the production of cell-associated TGF-alpha in differentiated keratinocytes which may function independent of its usual mitogenic effects.

Animals↗
PubMed2000

Overview of carcinogenesis: past, present and future.

In the foregoing articles, the editors of Carcinogenesis have identified the major themes of current carcinogenesis research and assembled an outstanding group of authors to review these areas. I have been asked to provide a historical overview of past accomplishments and describe how these contributed to the broader efforts to overcome the burden of human cancer. My assignment also included a look into the future. As scientists we formulate hypotheses that attempt to predict the future. Occasionally we are successful. The pioneers of carcinogenesis research were remarkably successful in predicting the future. Armed with primitive technology relative to today, these scientists studied the biology of carcinogenesis and conceptualized a framework for cancer pathogenesis that virtually everyone working in cancer research follows today. The current generation has been charged with filling in the details. In the details lay the future. Together with past accomplishments, these emerging details form a remarkable picture of progress in understanding and application, creating realistic and imminent promise to achieve victory in the fight against cancer.

Carcinogens↗
PubMed2000

Effect of a tyrosine 155 to phenylalanine mutation of protein kinase cdelta on the proliferative and tumorigenic properties of NIH 3T3 fibroblasts.

Tyrosine phosphorylation has emerged as an important mechanism in the regulation of enzyme function. In this paper, we describe a mutant of PKCdelta altered at a single tyrosine residue which has the opposite effect compared with wild-type PKCdelta on the growth characteristics of NIH 3T3 cells. Overexpression of wild-type PKCdelta results in a decreased growth rate and a lower cell density at confluency. On the other hand, overexpression of PKCdelta with a mutation from tyrosine to phenylalanine at position 155 results in a significantly higher rate of growth and a higher density at confluency compared with vector controls. Moreover, these cells are able to grow in soft agar and to form tumors in nude mice. In contrast to kinase negative PKC constructs, this mutant maintains in vitro kinase activity and shows a subcellular localization and a translocation pattern that are similar to those of the wild-type PKCdelta. Whether the altered biological effect is due to the missing phosphorylation on tyrosine or the mutation from tyrosine to phenylalanine per se remains under investigation.

3T3 Cells↗
PubMed2000

Frequent codon 12 Ki-ras mutations in mouse skin tumors initiated by N-methyl-N'-nitro-N-nitrosoguanidine and promoted by mezerein.

The skin tumor initiators N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 7,12-dimethylbenz[a]anthracene (DMBA) differ in effectiveness when tumor formation is promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Even at high doses, MNNG is less effective, producing fewer benign and malignant tumors with a longer latent period. In DMBA-initiated skin, 10 wk of TPA promotion produced a maximal tumor response. With MNNG, 20 wk of TPA promotion was required, producing nearly four times as many papillomas as 10 wk of promotion. Promotion of MNNG-initiated skin with mezerein induced the appearance of very rapidly-growing papillomas within 5 wk, 3 wk earlier than the first TPA-promoted papillomas. Thus, MNNG may induce a novel mutation resulting in a population of initiated cells that respond especially well to mezerein. Since ras mutations are common in experimental tumors in many tissues, we determined the frequency of activating mutations in the Ha-ras, Ki-ras, and N-ras oncogenes. Activating Ha-ras mutations were present in essentially all DMBA-initiated tumors and about 70% of MNNG-initiated tumors. No N-ras mutations were found in tumors lacking other ras mutations. Surprisingly, 41% of the papillomas arising in the first 11 wk in MNNG-initiated, mezerein-promoted mice bore mutations in codon 12 of the Ki-ras oncogene. Activating Ki-ras mutations were also found in more than 60% of squamous cell carcinomas and 40% of keratoacanthomas. Although mutations in Ha-ras are frequently detected in mouse skin tumors, mutations in Ki-ras are rare. This is the first report of mutated Ki-ras in skin tumors from mice initiated by MNNG.

Animals↗
PubMed1999

Defects in transforming growth factor-beta signaling cooperate with a Ras oncogene to cause rapid aneuploidy and malignant transformation of mouse keratinocytes.

Genetic inactivation of the transforming growth factor-beta (TGF-beta) signaling pathway can accelerate tumor progression in the mouse epidermal model of multistage carcinogenesis. By using an in vitro model of keratinocyte transformation that parallels in vivo malignant conversion to squamous cell carcinoma, we show that v-ras(Ha) transduced primary TGF-beta1-/- keratinocytes and keratinocytes expressing a TGF-beta type II dominant-negative receptor transgene have significantly higher frequencies of spontaneous transformation than control genotypes. Malignant transformation in the TGF-beta1-/- keratinocytes is preceded by aneuploidy and accumulation of chromosomal aberrations. Similarly, transient inactivation of TGF-beta signaling with a type II dominant-negative receptor adenovirus causes rapid changes in ploidy. Exogenous TGF-beta1 can suppress aneuploidy, chromosome breaks, and malignant transformation of the TGF-beta1-/- keratinocytes at concentrations that do not significantly arrest cell proliferation. These results point to genomic instability as a mechanism by which defects in TGF-beta signaling could accelerate tumor progression in mouse multistage carcinogenesis.

Aneuploidy↗
PubMed1999

p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein.

A novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53+/+ and p53-/- mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs from p53-/- mice. mc3s5/mtCLIC mRNA and protein are higher in p53+/+ compared with p53-/- basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor alpha also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor alpha.

Amino Acid Sequence↗
PubMed1999

Multiorgan transplacental and neonatal carcinogenicity of 3'-azido-3'-deoxythymidine in mice.

The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is used successfully for reduction of perinatal viral transmission. However toxic side effects including carcinogenesis are possible. To test this, pregnant CD-1 Swiss mice were given 25.0 or 12.5 mg AZT on gestation days 12-18. Previously we reported an increase in lung, liver, and female reproductive system tumors in offspring euthanized at 1 year (Olivero et al., J. Natl. Cancer Inst. 89, 1602-1608, 1997). Findings for all remaining offspring up to 2 years old are reported here. AZT effects were most prominent in female offspring, with a significant threefold increase in lung tumors, a reduction in lymphoblastic and follicle center cell lymphomas, and a significant increase in histiocytic sarcomas (0 in controls, 3% after low-dose AZT, and 8% after high-dose AZT, p = 0.022). Dose-dependent incidences of mammary gland, ovarian, and seminal vesicle tumors were low but significant: 0/106 controls, 3/105 low-dose, and 8/105 high-dose mice presented one of these neoplasms (p = 0.0025). Incidences of females showing any clearly AZT-related neoplasm, in lung, liver, ovary, or mammary gland or histiocytic sarcoma, in the second year, were 12/32 after the low dose and 14/27 after the high dose vs 3/23 controls (p = 0.0045). Also, the sensitivity of neonatal mice was assessed by administration of 25, 50, 100, or 200 mg/kg AZT on postnatal days 1 through 8. The effects at 2 years were similar to those seen after transplacental exposure, with significant increases in lung, liver, and mammary tumors in females. The results confirm that AZT is a moderately effective perinatal carcinogen in mice, targeting several tissue types.

Animals↗
PubMed1999

Genetic deletion of p21WAF1 enhances papilloma formation but not malignant conversion in experimental mouse skin carcinogenesis.

Tumor suppression by p53 is believed to reside in its ability to regulate gene transcription, including up-regulation of p21WAF1. In p53(-/-) mice, chemical- or oncogene-induced skin tumors undergo accelerated malignant conversion. To determine the contribution of the p21WAF1 gene product to epidermal carcinogenesis, animals +/+, +/-, and -/- for a null mutation in the p21WAF1 gene were treated once with 25 nmol 7,12-dimethylbenz[a]anthracene, followed by 5 microg of TPA two times/week for 20 weeks. Papilloma frequency was higher in the p21WAF1-deficient mice. However, the frequency of malignant conversion was similar among all three genotypes. After TPA treatment, all genotypes developed epidermal hyperplasia, although the labeling index was lower in p21WAF1 (-/-) epidermis compared with p21WAF1 (+/+). Furthermore, the expression of differentiation markers was the same across genotypes in untreated or TPA-treated epidermis. Similar frequencies of malignant conversion were also observed in an in vitro assay. Thus, p21WAF1 suppresses early stages of papilloma formation but not malignant progression in mouse skin carcinogenesis, and decreased levels of p21WAF1 do not account for the enhanced malignant conversion of p53 null epidermal tumors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1999

CRE DNA binding proteins bind to the AP-1 target sequence and suppress AP-1 transcriptional activity in mouse keratinocytes.

Previously, we have shown that nuclear extracts from cultured mouse keratinocytes induced to differentiate by increasing the levels of extra-cellular calcium contain Fra-1, Fra-2, Jun B, Jun D and c-Jun proteins that bind to the AP-1 DNA binding sequence. Despite this DNA binding activity, AP-1 reporter activity was suppressed in these cells. Here, we have detected the CREB family proteins CREB and CREMalpha as additional participants in the AP-1 DNA binding complex in differentiating keratinocytes. AP-1 and CRE DNA binding activity correlated with the induction of CREB, CREMalpha and ATF-1 and CREB phosphorylation at ser133 (ser133 phospho-CREB) in the transition from basal to differentiating keratinocytes, but the activity of a CRE reporter remained unchanged. In contrast, the CRE reporter was activated in the presence of the dominant-negative (DN) CREB mutants, KCREB and A-CREB, proteins that dimerize with CREB family members and block their ability to bind to DNA. The increase in CRE reporter activity in the presence of these mutants suggests that CRE-mediated transcriptional activity is suppressed in keratinocytes through protein-protein interactions involving a factor that dimerizes with the CREB leucine zipper. In experiments where the A-CREB mutant was co-transfected with an AP-1 reporter construct, transcriptional activity was also increased indicating that a CREB family member binds AP-1 sites and represses AP-1 transcriptional activity as well. Exogenous expression of the transcriptional repressor CREMalpha down-regulated both CRE and AP-1 reporters in keratinocytes suggesting that this factor may contribute to the suppression of AP-1 transcriptional activity observed in differentiating keratinocytes.

Animals↗
PubMed1999

Expression of interferon-beta is associated with growth arrest of murine and human epidermal cells.

The cytokine interferon-beta is a regulator of cell replication and function, including invasion and induction of angiogenesis. The goal of this study was to determine whether the expression of interferon-beta by cells in the epidermis correlated with terminal differentiation. In situ hybridization analysis and immunohistochemical staining of formalin-fixed, paraffin-embedded specimens of normal human and murine epidermis and human and murine skin tumors of epithelial origin revealed that only differentiated, nondividing cells of the epidermis expressed interferon-beta protein. Keratinocyte cultures established from the epidermis of 3 d old mice were maintained under conditions permitting continuous cell division or induction of differentiation. Continuously dividing cells did not produce interferon-beta whereas nondividing differentiated cells expressing keratin 1 did. Growth-arrested, undifferentiated keratinocytes also expressed interferon-beta protein. Neutralizing interferon-beta in the culture medium inhibited differentiation, but the addition of exogenous interferon-beta did not stimulate differentiation. These data indicate that interferon-beta is produced by growth-arrested, terminally differentiated keratinocytes.

Animals↗
PubMed1999

Protein kinase Cdelta targets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector.

Inactivation of protein kinase Cdelta (PKCdelta) is associated with resistance to terminal cell death in epidermal tumor cells, suggesting that activation of PKCdelta in normal epidermis may be a component of a cell death pathway. To test this hypothesis, we constructed an adenovirus vector carrying an epitope-tagged PKCdelta under a cytomegalovirus promoter to overexpress PKCdelta in normal and neoplastic keratinocytes. While PKCdelta overexpression was detected by immunoblotting in keratinocytes, the expression level of other PKC isozymes, including PKCalpha, PKCepsilon, PKCzeta, and PKCeta, did not change. Calcium-independent PKC-specific kinase activity increased after infection of keratinocytes with the PKCdelta adenovirus. Activation of PKCdelta by 12-O-tetradecanoylphorbol-13-acetate (TPA) at a nanomolar concentration was lethal to normal and neoplastic mouse and human keratinocytes overexpressing PKCdelta. Lethality was inhibited by PKC selective inhibitors, GF109203X and Ro-32-0432. TPA-induced cell death was apoptotic as evidenced by morphological criteria, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, DNA fragmentation, and increased caspase activity. Subcellular fractionation indicated that PKCdelta translocated to a mitochondrial enriched fraction after TPA activation, and this finding was confirmed by confocal microscopy of cells expressing a transfected PKCdelta-green fluorescent protein fusion protein. Furthermore, activation of PKCdelta in keratinocytes altered mitochondrial membrane potential, as indicated by rhodamine-123 fluorescence. Mitochondrial inhibitors, rotenone and antimycin A, reduced TPA-induced cell death in PKCdelta-overexpressing keratinocytes. These results indicate that PKCdelta can initiate a death pathway in keratinocytes that involves direct interaction with mitochondria and alterations of mitochondrial function.

Adenoviridae↗