Armand Hornia - Academia.edu (original) (raw)

Papers by Armand Hornia

Molecular and Cellular Biology, Feb 1, 1998

Treatment of cells with tumor-promoting phorbol esters results in the activation but then depleti... more Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms ␣, ␦, and in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC ␦. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC ␣, ␦, and were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA-and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC ␣ could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway.

Journal of Cellular and Molecular Medicine, Mar 4, 2008

This article cites 44 articles, 17 of which can be accessed free

Investigative Ophthalmology & Visual Science, 2006

Cell Growth Differentiation the Molecular Biology Journal of the American Association For Cancer Research, Apr 1, 2001

The tumor-promoting phorbol ester TPA (12-Otetradecanoylphorbol-13-acetate) cooperates with c-Src... more The tumor-promoting phorbol ester TPA (12-Otetradecanoylphorbol-13-acetate) cooperates with c-Src overexpression to transform rat fibroblasts. TPA transforms c-Src-overexpressing cells by depleting the ␦ isoform of protein kinase C (PKC␦). Tamoxifen, which has both estrogen-mimetic and estrogen-antagonist properties, has been widely used to improve the prognosis of breast cancer patients. However, with extended use, there is an increased risk for endometrial and other cancers that can be observed within 10 years of treatment. We report here that tamoxifen, similar to TPA, cooperates with c-Src overexpression to transform 3Y1 rat fibroblasts. Tamoxifen induced both DNA synthesis and anchorage-independent cell proliferation in c-Srcoverexpressing, but not in parental, 3Y1 rat fibroblasts. Tamoxifen also induced an association between c-Src and PKC␦ that resulted in the tyrosine phosphorylation and down-regulation of PKC␦. These phenotypes were not induced by estrogen, indicating that the effect of tamoxifen was in addition to any estrogen-mimetic effects. Thus, in addition to the hyperplasia-inducing capability of an estrogen-mimetic, tamoxifen has an additional tumor-promoting capability similar to that of TPA. The dual tumor-promoting capability of both estrogen-and TPA-mimetic properties for tamoxifen may contribute to the increased incidence of endometrial cancers observed in the relatively short exposure period of <10 years.

Molecular and Cellular Biology, 1999

Downregulation of protein kinase C δ (PKC δ) by treatment with the tumor-promoting phorbol ester ... more Downregulation of protein kinase C δ (PKC δ) by treatment with the tumor-promoting phorbol ester 12- O -tetradecanoylphorbol-13-acetate (TPA) transforms cells that overexpress the non-receptor class tyrosine kinase c-Src (Z. Lu et al., Mol. Cell. Biol. 17:3418–3428, 1997). We extended these studies to cells overexpressing a receptor class tyrosine kinase, the epidermal growth factor (EGF) receptor (EGFR cells); like c-Src, the EGF receptor is overexpressed in several human tumors. In contrast with expectations, downregulation of PKC isoforms with TPA did not transform the EGFR cells; however, treatment with EGF did transform these cells. Since TPA downregulates all phorbol ester-responsive PKC isoforms, we examined the effects of PKC δ- and PKC α-specific inhibitors and the expression of dominant negative mutants for both PKC δ and α. Consistent with a tumor-suppressing function for PKC δ, the PKC δ-specific inhibitor rottlerin and a dominant negative PKC δ mutant transformed the EG...

The Journal of biological chemistry, Jan 22, 2005

Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including corn... more Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including corneaspecific keratocan. On plastic dishes, human, bovine and rabbit keratocytes lose their characteristic dendritic morphology and keratocan expression when cultured in serum-containing media. Herein, we demonstrated that murine keratocytes also acquired a fibroblastic shape and lost keratocan expression after first passage when cultured on plastic in the presence of serum. In contrast, cells expanded on human amniotic membrane (AM) stromal matrix maintained a 3-dimensional dendritic morphology and expressed keratocan mRNA and protein for at least 8 passages before senescence. When keratocytes were cultured on AM, the promoter activity of TGF-β2 and TGF-βRII was downregulated as compared to that on plastic. Furthermore, cells on AM continuously retained Smad 2 and Smad 4 in the cytoplasm and did not express α-SMA even when 10 ng/ml TGF-β1 was added in a serum-free medium for up to 5 days. In parallel to such downregulation of TGF-β signaling, keratocan promoter-driven ECFP expression was observed in cells cultured either on AM in the presence of serum, or on plastic containing serum treated with a neutralizing antibody to TGF-β. Collectively, these results indicate that downregulation of Smad-mediated TGF-β signaling is an important mechanism for cultured keratocytes to maintain a normal phenotype while continuously expanded in a serum-containing medium. This strategy of suppressing TGF-β signaling, achieved by AM stromal matrix in part via suppression of TGF-β gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.

The tumor-promoting phorbol ester TPA (12-O- tetradecanoylphorbol-13-acetate) cooperates with c- ... more The tumor-promoting phorbol ester TPA (12-O- tetradecanoylphorbol-13-acetate) cooperates with c- Src overexpression to transform rat fibroblasts. TPA transforms c-Src-overexpressing cells by depleting the d isoform of protein kinase C (PKCd). Tamoxifen, which has both estrogen-mimetic and estrogen-antagonist properties, has been widely used to improve the prognosis of breast cancer patients. However, with extended use, there is an increased risk for

Downregulation of protein kinase C ␦ (PKC ␦) by treatment with the tumor-promoting phorbol ester ... more Downregulation of protein kinase C ␦ (PKC ␦) by treatment with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) transforms cells that overexpress the non-receptor class tyrosine kinase c-Src (Z. Lu et al., Mol. Cell. Biol. 17:3418-3428, 1997)

Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with p... more Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced downregulation of the PKC␦ isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKC␦ as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKC␦ mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKC␦, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKC␦, which has an apparent tumor suppressor function.

Journal of Cellular and Molecular Medicine, 2008

The limbal region of the adult cornea contains stem cells which are ultimately responsible for re... more The limbal region of the adult cornea contains stem cells which are ultimately responsible for regeneration of the corneal epithelium during wound repair. However, primarily-isolated murine corneal/limbal epithelial cells rapidly senesce on plastic in a serum-free low [Ca 2+ ] medium, suggesting only transit amplifying cells are promoted. We developed a novel expansion method by seeding at a low cell density (<500 cells/cm 2 ) and prolonging each culture time beyond the lifespan of transit amplifying cells (4 weeks). Expanded cells were uniformly small, negative to K12 keratin, but positive for p63 nuclear staining, and could be subcultured beyond 100 passages. After limiting dilution, one clone (TKE2) was selected that exhibited single cell clonal expansion with a doubling time of 34.2 hrs, and had normal karyotyping, but no anchorage-independent growth. A single cell could be continually expanded to a confluent monolayer on denuded amniotic membrane and became stratified by exposing to the air-medium interface. The resultant stratified epithelium expressed K14 keratin, involucrin, connexin 43 and p63, but not K12 keratin or Pax 6. However, expression of K12 could be up-regulated by increasing extracellular calcium concentration and addition of foetal bovine serum (FBS) at P12, but less so at P85. Therefore, this murine limbal/corneal epithelium-derived progenitor cell line still retained the plasticity for adopting corneal lineage differentiation, could be useful for investigating limbal niche cues that may promote corneal epithelial fate decision.

Molecular and Cellular Biology, 2000

3Y1 rat fibroblasts overexpressing the epidermal growth factor (EGF) receptor (EGFR cells) become... more 3Y1 rat fibroblasts overexpressing the epidermal growth factor (EGF) receptor (EGFR cells) become transformed when treated with EGF. A common response to oncogenic and mitogenic stimuli is elevated phospholipase D (PLD) activity. RalA, a small GTPase that functions as a downstream effector molecule of Ras, exists in a complex with PLD1. In the EGFR cells, EGF induced a Ras-dependent activation of RalA. The activation of PLD by EGF in these cells was dependent upon both Ras and RalA. In contrast, EGF-induced activation of Erk1, Erk2, and Jun kinase was dependent on Ras but independent of RalA, indicating divergent pathways activated by EGF and mediated by Ras. The transformed phenotype induced by EGF in the EGFR cells was dependent upon both Ras and RalA. Importantly, overexpression of wild-type RalA or an activated RalA mutant increased PLD activity in the absence of EGF and transformed the EGFR cells. Although overexpression of PLD1 is generally toxic to cells, the EGFR cells not only tolerated PLD1 overexpression but also became transformed in the absence of EGF. These data demonstrate that either RalA or PLD1 can cooperate with EGF receptor to transform cells.