SV40-transformed Human Keratinocytes Research Articles

Cystatin A suppresses ultraviolet B-induced apoptosis of keratinocytes

Cystatin A is a cysteine proteinase inhibitor abundantly expressed in keratinocytes. Although cystatin A is one of the cornified cell envelope constituents and expressed in the upper epidermis, its precise function is still unknown. Ultraviolet B irradiation (UVB) induces apoptosis accompanied with the activation of cysteine proteinases, caspases. We investigated the effect of cystatin A on UVB-induced apoptosis of keratinocytes. We assessed the caspase activities and apoptotic cell numbers induced by UVB ittadiation in cystatin A gene transfected keratinocytes. UVB-induced pro-caspase 3 cleavage and caspase 3 activation were suppressed in cystatin A expression vector-transfected SV40-transformed human keratinocytes (SVHK). Furthermore, the transfected SVHK cells were resistant to UVB-induced apoptosis. In contrast neither caspase 8 nor caspase 9 activities were affected by UVB irradiation in cystatin A-transfected SVHK cells. The effects were also observed in cystatin A expression adenovirus vector-transfected cultured normal human keratinocytes (NHK). Conversely knockdown of cystatin A by si-RNA induced marked apoptosis of NHK cells following UVB irradiation accompanied with increased caspase 3 activity. In order to confirm the antiapoptotic effect of cystatin A in vivo UVB irradiation was performed on cystatin A transgenic mice (cystatin A-tg). The epidermis from cystatin A-tg was resistant to UVB-induced apoptosis compared to control mice epidermis. These results indicate that cystatin A suppresses UVB-induced apoptosis of keratinocytes by the inhibition of caspase 3 activation.

Ultraviolet B irradiation increases keratin 5 and keratin 14 expression through epidermal growth factor receptor of SV40-transformed human keratinocytes.

Keratin intermediate filaments are heteropolymers composed of type I and type II keratins. Ultraviolet B (UVB) irradiation induces keratin expression by keratinocytes. Using SV40-transformed human keratinocytes (SVHK), we investigated the effect of UVB irradiation on keratin expression. UVB irradiation (10 mJ/cm(2)) increased keratin 5 and keratin 14 mRNAs and proteins without affecting cell viability. Upregulation of keratin 5 and keratin 14 was dependent on the dose of radiation: the effect was observed at 5 mJ/cm(2) and the maximal effect was observed at 10 mJ/cm(2). Higher UVB doses (more than 10 mJ/cm(2)) were cytotoxic. Expression of keratin 1 and keratin 10 was marginal in SVHK and was not affected at either the mRNA or protein level by UVB. The stimulatory effects on keratin 5 and keratin 14 expression were also observed in cultured normal human keratinocytes (NHK) and HaCaT keratinocytes. The tyrosine kinase inhibitor, genistein, and the epidermal growth factor (EGF) receptor inhibitor, AG1429, significantly suppressed the increase in expression of keratin 5 and keratin 14 by SVHK. In contrast, the suppressive effect was not observed with the protein kinase C inhibitor, H-7. Furthermore, pretreatment with neutralizing anti-EGF receptor antibody also suppressed UVB-induced keratin 5 and keratin 14 expression by SVHK, NHK and HaCaT cells. UVB irradiation did not affect the steady-state expression of TGF-alpha by SVHK. Immunoprecipitation and immunohistochemical studies revealed that UVB irradiation induced EGF receptor activation in the absence of EGF and TGF-alpha. These results indicate that UVB increases keratin 5 and keratin 14 expression through direct activation of the EGF receptor in SVHK.

Ultraviolet B irradiation induces apoptosis of keratinocytes by direct activation of Fas antigen.

Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes, where p53 has been suggested to play an important role. Recently we have shown that UVB irradiation induces apoptosis of SV40-transformed human keratinocytes (SVHK cells). Because p53 function is impaired in SVHK cells by large T antigen, a UVB-induced p53-independent apoptotic pathway was suggested. We investigated the UVB-induced apoptotic pathway using various keratinocytes. Cultured mouse keratinocytes of homozygous p53 deficient mice (p53(-/-)) were markedly resistant to UVB-induced apoptosis compared with keratinocytes from wild or heterozygous p53 deficient mice (p53(-/+)). Twenty per cent of keratinocytes derived from p53 (-/-) mice, however, induced apoptosis following UVB irradiation. Analysis using caspase inhibitors disclosed activation of caspase 8 and 3 in UVB-irradiated SVHK cells. Keratinocytes derived from MRL/lpr mice, which have mutated Fas antigen, showed diminished UVB-induced apoptosis suggesting that Fas antigen is significantly involved in UVB-induced apoptosis. Immunohistochemical analysis revealed that UVB irradiation induces aggregation of Fas antigen showing a dense dot-like staining, which was also observed in SVHK cells treated with agonistic anti-Fas antibody, CH11. Pretreatment of antagonistic anti-Fas antibody, ZB4, inhibited CH11-induced but not UVB-induced multimerization of Fas antigen. Furthemore, UVB irradiation did not affect the basal expression of Fas ligand mRNA, protein and soluble Fas ligand. These results indicate that UVB irradiation induces multimerization of Fas antigen that results in apoptosis without the Fas ligand.

Differential phosphorylation of mitogen-activated protein kinase families by epidermal growth factor and ultraviolet B irradiation in SV40-transformed human keratinocytes

SV-40 transformed human keratinocytes (SVHK cells) were stimulated with epidermal growth factor (EGF) and ultraviolet B (UVB) irradiation. Following the stimulation, cell growth, apoptosis, and the activities of mitogen-activated protein (MAP) kinase families were analyzed. EGF (100 ng/ml) increased SVHK cell number compared with control cells cultured in serum-free DMEM medium. The EGF-stimulated cells did not show DNA fragmentation. In contrast, UVB irradiation (40 mJ/cm 2) markedly decreased viable cell number that was accompanied with DNA fragmentation. EGF stimulated extracellular signal-regulated kinase (ERK) and stress-activated protein kinase/c-Jun N-terminal kinase (JNK). Following the EGF stimulation, phosphorylated ERK and JNK were detected by phospho-p42/44 MAP kinase antibody and phospho-SAPK/JNK antibody, respectively. On the other hand, UVB irradiation stimulated the phosphorylation of p38 and JNK but not of ERK. The stimulation of ERK and JNK induced by EGF was observed earlier than the stimulation of p38 and JNK induced by UVB. PD98059, a specific MAP kinase kinase (MAPKK) 1 (also referred to as MEK1) inhibitor, inhibited EGF-dependent cell proliferation, that was associated with the inhibition of ERK and JNK phosphorylation. In contrast, UVB-induced overall cell death was not significantly affected by PD98059, that inhibited phosphorylation of JNK but not of p38. PD98059, however, significantly augmented UVB-induced cell death earlier time points (30 min–2 h). These results indicate that ERK and JNK are activated following EGF stimulation that might be associated with cell proliferation. On the other hand, UVB-induced apoptosis seems to be mostly associated with the activation of p38. JNK stimulation might provide an anti-apoptotic tonus during the UVB-induced, p38-associated SVHK cell death.

Copper, zinc–superoxide dismutase protects from ultraviolet B-induced apoptosis of SV40-transformed human keratinocytes: the protection is associated with the increased levels of antioxidant enzymes

It has been reported that cellular oxidative stress induces apoptosis. Ultraviolet radiation that generates reactive oxygen intermediates (ROIs) also induces apoptosis. Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Mammalian cells express two isozymes of SOD, copper, zinc–SOD (Cu, Zn–SOD) and manganese–SOD (Mn–SOD). Using SV40-transformed human keratinocytes (SVHK cells), we investigated the role of SODs in the ultraviolet B (UVB) irradiation-induced apoptosis. UVB irradiation decreased transiently Cu, Zn– and Mn–SOD activities and their protein levels, with subsequent recovery to the basal levels by 24 h. The UVB-induced decrease in SOD activity was dose-dependent and the maximal effect was obtained at 75 mJ/cm 2. The decrease in Cu, Zn–SOD was more marked than that in Mn–SOD. The cell death assay, annexin-V/propidium iodide flow cytometry, and DNA fragmentation analysis revealed that UVB irradiation induces apoptosis in SVHK cells. The UVB-induced apoptosis was suppressed by the treatment of antioxidants, catalase, glutathione, and α-tochopherol. The stable transfection of Cu, Zn–SOD expression vectors into SVHK cells was accompanied by the increased activities of antioxidant enzymes, catalase, and glutathione reductase, as well as glutathione and the cells were shown to be more resistant to UVB-induced apoptosis. In contrast, the transfection of Mn–SOD affected neither activities of antioxidant enzymes nor the UVB-induced apoptosis. The transfection of Cu, Zn–SOD antisense oligomers but not sense oligomers into SVHK or Cu, Zn–SOD cDNA-transfected SVHK (C2) cells significantly decreased the antioxidant enzyme activities and increased the UVB-induced apoptosis. On the other hand, the transfection of Mn–SOD antisense oligomers did not affect the UVB-induced apoptosis. These results suggest that the transfection of Cu, Zn–SOD expression vector, which is accompanied by the increased level of antioxidant enzymes, suppresses the UVB-induced apoptosis of SVHK cells.

Fas Antigen Modulates Ultraviolet B-Induced Apoptosis of SVHK Cells: Sequential Activation of Caspases 8, 3, and 1 in the Apoptotic Process

Interferon-γ (IFN-γ) induces various apoptosis-related proteins, including Fas antigen (Fas) in keratinocytes. Ultraviolet B (UVB) irradiation produces “sunburn cells,” a specific type of apoptosis. Previously, we reported that IFN-γ augments Fas-dependent apoptosis of SV40-transformed human keratinocytes (SVHK cells). Caspases are a new class of cysteine proteinases that play an important role in apoptosis. We investigated the mechanism of UVB-induced apoptosis by examining activation of the caspase cascade. UVB irradiation of SVHK cells increased the activities of caspases 1, 3, and 8, which were detected at 3 h, and peak activities occurred at 6 h. Pretreatment of SVHK cells with IFN-γ significantly increased the activity of caspases 1, 3, and 8. UVB-induced caspase 8 stimulation was significantly suppressed only by caspase 8 inhibitor, while inhibitors of caspases 1, 3, and 8 significantly suppressed UVB-induced caspase 1 stimulation. Caspase 3 and 8 inhibitors, but not caspase 1 inhibitor, significantly suppressed UVB-induced caspase 3 activity, suggesting sequential activation of caspases 8, 3, and 1 in UVB-irradiated SVHK cells. Cross-linking and immunoprecipitation analyses showed multimerization of Fas antigen following UVB irradiation of SVHK cells. Pretreatment of SVHK cells with IFN-γ significantly augmented UVB-induced apoptosis that was accompanied by increased Fas expression. The susceptibility to UVB-induced apoptosis was also increased in Fas-transfected SVHK cells (F2 cells). Neutralizing anti-Fas antibody significantly suppressed caspase activation and Fas-dependent apoptosis of SVHK cells and F2 cells. In contrast, UVB-induced caspase activation and apoptosis were not inhibited by neutralizing anti-Fas antibody in both cell lines. Our results suggest that UVB directly activates Fas and subsequent caspase cascade resulting in apoptosis of SVHK cells. Furthermore, the expression level of Fas antigen in keratinocytes influenced their susceptibility to UVB-induced apoptosis.

Structure and Transcriptional Regulation of the Human Cystatin A Gene: THE 12-O-TETRADECANOYLPHORBOL-13-ACETATE (TPA) RESPONSIVE ELEMENT-2 SITE (−272 TO −278) ON CYSTATIN A GENE IS CRITICAL FOR TPA-DEPENDENT REGULATION

Cystatin A, a cysteine proteinase inhibitor, is one of the precursor proteins of cornified cell envelope of keratinocytes and is expressed during the late stage of keratinocyte differentiation. We have isolated and characterized the human cystatin A gene. The cystatin A gene consists of three exons and two introns. The first, the second, and the third exons consist of coding sequences that are 66, 102, and 126 base pairs in length, respectively. The first and the second introns consist of 14 and 3.6 kilobase pairs, respectively. The transcription initiation site was located 55 base pairs upstream from the first translation site. The fragment, +77 to -2595 in the 5'-flanking region of the human cystatin A gene, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector, p2672CAT, produced a significant CAT activity in transiently transfected SV40-transformed human keratinocytes (SVHK cells), that were further stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator. Sequence analysis of the gene detected three TPA responsive elements (TRE-1, TRE-2, and TRE-3) and one AP-2 site on the 5' upstream promoter region. Deletion analyses of the p2672CAT vector demonstrated that TRE-2, which was located between -272 and -278, was critical for the regulation by TPA. Gel shift analyses revealed that c-Jun, JunD, and c-Fos bound to the TRE-2 region and that the p2672CAT activity level was elevated by co-transfection with c-Jun and c-Fos or with JunD and c-Fos expression vectors. Furthermore, co-transfection of SVHK cells with the protein kinase C-alpha expression vector and the p2672CAT expression vector also resulted in an increased CAT activity. These results indicate that the 5'-flanking region of the human cystatin A gene confers promoter activity and contains a TRE (TRE-2) that mediates, at least in part, the enhanced expression of this gene by TPA.

Interferon- γ-dependent induction of manganese superoxide dismutase activity of SV40-transformed human keratinocytes by anti-Fas antibody and by TNF- α

It has been reported that cellular oxidative stress induces apoptosis, that may be inhibited by scavengers of reactive oxygen intermediates (ROIs). Superoxide dismutase (SOD) is among the most active scavengers of ROIs, providing defense against the cellular oxidative stress. Fas antigen and tumor necrosis factor (TNF) receptor are the cell surface proteins, stimulation of which induces apoptosis of keratinocytes. Using SV40-transformed human keratinocytes (SVHK cells), we investigated the effects of anti-Fas antibody and TNF- α on the SOD activity. Treatment of SVHK cells with anti-Fas antibody or TNF- α in the presence of interferon- γ (IFN- γ) resulted in an increase in Mn-SOD activity, Cu,Zn-SOD activity was not affected. In the absence of IFN- γ, no increase in Mn-SOD activity was detected. The induction of IFN- γ-dependent Mn-SOD activity by anti-Fas antibody or TNF- α was concentration-dependent; the maximal effect was observed at 1–10 μg/ml and 5–10 ng/ml, respectively. The increase in Mn-SOD activity was observed at 6 h following the treatment and remained for at least 48 h. Northern blot analyses showed that Mn-SOD mRNA increased within 3 h without a significant change in Cu,Zn-SOD mRNA. The addition of both anti-Fas antibody and TNF- α in the presence of IFN- γ resulted in an additive increase in Mn-SOD activity. Although the addition of 12- o-tetradecanoylphorbol-13-acetate (TPA) singly to the incubation medium had no effect on either Mn-, or Cu,Zn-SOD activity, it significantly augmented the IFN- γ-dependent induction of Mn-SOD activity by anti-Fas antibody or by TNF- α. The protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7), significantly inhibited the TPA-dependent increase in Mn-SOD activity. These results indicate that the stimulation of Fas antigen or TNF receptor increases Mn-SOD activity of SVHK cells in the presence of IFN- γ and that TPA augments the process through the activation of protein kinase C.

The α and η Isoforms of Protein Kinase C Stimulate Transcription of Human Involucrin Gene

Involucrin is one of the precursor proteins of the cornified cell envelope that is formed beneath the cell membrane during terminal differentiation of keratinocytes. 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a potent protein kinase C (PKC) activator, induces terminal differentiation of keratinocytes. We previously demonstrated that involucrin promoter activity is stimulated by TPA in cultured fetal rat skin keratinocytes. PKC is a large family of proteins and keratinocytes containing five PKC isozymes: alpha, delta, epsilon, eta, and zeta. In order to determine the role of the PKC isozyme(s) on involucrin gene expression, we constructed the chloramphenicol acetyl transferase (CAT)-involucrin promoter expression vector by connecting the 5'-upstream region of the human involucrin gene containing the untranslated first exon to the CAT reporter gene. The CAT-involucrin promoter expression vector was transfected with various PKC isozyme expression vectors into SV40-transformed human keratinocytes (SVHK cells). Transfection of the CAT-involucrin promoter expression vector with PKC-alpha or PKC-eta expression vectors resulted in a significant increase in the TPA-dependent involucrin promoter activity. The PKC inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride, inhibited the promoter activity stimulated by TPA. Transfection of PKC-delta, -epsilon, and -zeta had no effect on the involucrin-promoter activity. Although the promoter activity was stimulated by transfection of PKC-gamma, TPA did not enhance the promoter activity in the PKC-gamma-transfected SVHK cells. Previously we showed three AP-1 binding sites (AP1-1, -2, and -3) on the involucrin promoter region. Both the basal and the TPA-stimulated involucrin promoter activities were suppressed by deleting the AP1-1 site (-119 to -113) that is the most proximal to the transcription start site. The deletion of AP1-2 (-297 to -303) or AP1-3 (-447 to -453) did not affect the involucrin promoter activity. Gel retardation analyses disclosed that TPA stimulated the specific DNA binding of the nuclear protein(s) of control, PKC-alpha, or PKC-eta-transfected SVHK cells, but not of PKC-gamma-transfected cells. Addition of anti-c-Jun and anti-c-Fos antibodies decreased the specific protein-DNA complex band with a concomitant appearance of supershifted bands. These results indicate that PKC, specifically PKC-alpha and PKC-eta, mediates the TPA-dependent activation of involucrin gene expression of SVHK cells. PKC-gamma, which is not present in keratinocytes, also induces involucrin gene expression in a TPA-independent manner, when introduced into SVHK cells.

Interleukin-1β-Converting Enzyme and CPP32 Are Involved in Ultraviolet B-Induced Apoptosis of SV40-Transformed Human Keratinocytes

Interleukin-1β-converting enzyme (ICE) and CPP32 are cysteine proteinases, that are involved in apoptotic process in various cell systems. We investigated the effects of ICE on ultraviolet B (UVB) induced-apoptosis in SV40-transformed human keratinocytes (SVHK cells). The ICE inhibitor (Z-Val-Ala-Asp-CH2F) and CPP32 inhibitor (Z-Asp-Glu-Val-Asp-CH2F) blocked the apoptotic cell death caused by UVB irradiation. The addition of both ICE and CPP32 inhibitors to the incubation medium resulted in neither an additive nor a synergistic suppression of UVB-induced apoptosis. Reverse transcription and polymerase chain reaction (RT-PCR) analysis indicated that SVHK cells expressed ICE-α, and β mRNAs. UVB irradiation increased the mRNA of both isoforms and Western blot analysis confirmed that UVB increased an active form of ICE protein, p20, that is generated by autoproteolytic cleavage of inactive 45 kDa proenzyme derived from both mRNAs. Transfection of ICE expression vector into SVHK cells resulted in apoptosis in a dose dependent manner and UVB-irradiation further augmented the ICE expression vector-induced apoptosis. These results indicate that ICE plays an important role in UVB-induced apoptosis of SVHK cells.

Cloning of Human Keratolinin cDNA: Keratolinin Is Identical with a Cysteine Proteinase Inhibitor, Cysteine A, and Is Regulated by Ca2+, TPA, and cAMP

Keratolinin has been described as one of the precursor proteins of cornified cell envelope of keratinocytes. Using rabbit polyclonal anti-human keratolinin antibody, we isolated a cDNA clone of human keratolinin gene from a human Agt11 cDNA expression library that was constructed by random priming from poly(A)+RNA extracted from cultured normal human keratinocytes. Screening by rabbit anti-human keratolinin antibody detected one positive clone (HKL-1). The recombinant 12.5-kDa protein constructed from the clone reacted specifically with the anti-human keratolinin antibody. DNA sequence analysis revealed that HKL-1 clone was 448 bp long, and its putative amino acid sequence was identical with that of a human cysteine proteinase inhibitor, cystatin A. Western blot analysis showed that the commercially available recombinant cystatin A also reacted specifically with the anti-human keratolinin antibody. Northern blot analysis indicated that HKL-1 clone hybridizes with mRNA of about 0.5 kb, consistent with the size of the HKL-1 clone. The keratolinin mRNA was highly expressed in cultured human keratinocytes in high Ca2+ (1 mM); in low Ca2+ (0.05 mM), the keratolinin mRNA expression was significantly lower. Using SV40-transformed human keratinocytes (SVHK cells), we further analyzed the regulation of keratolinin mRNA. In low Ca2+ (0.05 mM), keratolinin mRNA in SVHK cells was marginally detectable. Upon shift to 1 mM calcium, keratolinin mRNA was markedly increased. The upregulation of keratolinin mRNA was also observed by the treatment of SVHK cells with 10 ng TPA per ml or 100 microM forskolin under low calcium conditions (0.05 mM). Our results indicate that keratolinin is identical with cystatin A, a cysteine proteinase inhibitor, and its expression is positively regulated by Ca2+, TPA, and forskolin.

DMSO induces apoptosis in SV40-transformed human keratinocytes, but not in normal keratinocytes.

We found that dimethyl-sulfoxide (DMSO) at concentrations of 2.5% induced apoptosis in SV40-immortalized human keratinocytes, while normal keratinocytes were arrested at the boundary of G1/S phase under the same conditions. DMSO-induced apoptosis in SV-40 immortalized keratinocytes was not associated with change in phosphorylated state of the retinoblastoma susceptibility gene. When SV40-immortalized cells were treated with 2.5% DMSO, dissociation of the complex was observed by immunoblotting of SV40 T antigen from immunoprecipitated p53 protein fraction.

Interferon-γ-Dependent Stimulation of Fas Antigen in SV40-Transformed Human Keratinocytes: Modulation of the Apoptotic Process by Protein Kinase C

Fas antigen is a cell membrane protein that has been suggested to mediate apoptosis. Using SV40-transformed human keratinocytes, we investigated the Fas-antigen-dependent apoptotic process. The expression of Fas antigen mRNA was markedly induced by interferon-gamma (IFN-gamma) treatment (500 U/ml). After IFN-gamma treatment in the presence of anti-Fas monoclonal antibody, apoptosis was induced, as detected by the formation of nucleosome-sized fragments of DNA and morphologically by apoptotic cells with round homogeneous nuclear beads detected by acridine orange staining. The apoptotic SV40-transformed keratinocytes were analyzed quantitatively by enzyme-linked immunosorbent assay using antihistone and peroxidase-conjugated anti-DNA antibodies to detect cell death. The IFN-gamma- and anti-Fas antibody-dependent apoptotis was observed by 3 h, and the maximal response was observed by 12 h. The induction of apoptosis was significantly augmented by treatment with 10 ng/ml 12-o-tetradecanoyl-phorbol-13-acetate (TPA). TPA alone had no effect on either Fas antigen expression or on the apoptotic process. Other protein kinase C activators (1-oleoyl-2-acetylglycerol and mezerein) also stimulated IFN-gamma-dependent apoptosis, whereas 4-o-methyl phorbol myristate acetate, a very weak protein kinase C activator, had only a slight effect. The TPA-induced augmentation of apoptosis was inhibited by the protein kinase C inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7). However, H-7 inhibited only the TPA-induced augmentation of apoptosis; the basal IFN-gamma- and anti-Fas-dependent apoptosis remained in the presence of H-7. Northern blot analysis revealed that c-jun mRNA was induced by IFN-gamma plus anti-Fas antibody treatment as well as by TPA treatment; the addition of IFN-gamma alone to the incubation medium had no effect on the expression of c-jun mRNA. These results indicate that IFN-gamma induces a Fas-antigen-dependent apoptotic process in SV40-transformed keratinocytes and that TPA augments the process through the activation of protein kinase C.

The levels of ubiquitinated histone H2A are highly upregulated in transformed human cells: Partial colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase

The levels and distribution of ubiquitinated histone H2A (uH2A) have been studied in normal and transformed human cells using a monoclonal antibody (mAb E6C5) that reacts specifically with this ubiquitin conjugate as determined by two-dimensional gel western blotting and microsequencing. Immunoblotting experiments demonstrated that the levels of the protein are highly upregulated in SV40-transformed human fibroblasts (WI-38 SV40) and keratinocytes (K14) relative to their normal counterparts, a finding that was further confirmed by indirect immunofluorescence studies of formaldehyde/Triton X-100-treated cells, which showed that about 97% of the transformed cells and 26% of the normal populations reacted with the antibody to yield a fine granular nuclear staining associated with the chromatin. Transformed cells contained in addition clusters of uH2A that were quite abundant and that showed variable size, shape and distribution even within a single cell line. The clusters, which were rare in normal cells, did not colocalize with other known nuclear antigens and may correspond to novel nuclear domains where ubiquitination/deubiquitination takes place. Electron microscopic immunocytochemistry of K14 cells confirmed the existence of the clusters. Double immunofluorescence studies of K14 keratinocytes with proliferating cell nuclear antigen (PCNA)/cyclin antibodies, which react with the nuclei of cells engaged in DNA replication, showed partial colocalization of PCNA/cyclin foci and large uH2A clusters in about 14% of the S-phase cells, and these corresponded mainly to late S-phase cells. Inhibition of DNA replication with hydroxyurea resulted in an overall increase in the intensity of the uH2A staining as well as in a more clear colocalization of uH2A clusters and PCNA/cyclin foci. Taken together, the results support the contention that uH2A plays a role at some stage of DNA replication.

Repression of involucrin gene expression by transcriptional enhancer factor 1 (TEF-1).

Involucrin is one of the precursor proteins of keratinocyte cornified envelope that is formed beneath the inner surface of the cell membrane during terminal differentiation. Although involucrin is specifically expressed in the upper squamous cells of the epidermis, the precise regulatory mechanism of involucrin gene expression remains unknown. Transcriptional enhancer factor 1 (TEF-1), which binds to SV40 enhancer, is a nuclear protein expressed in various types of cells including keratinocytes. Immunohistochemical study has revealed that TEF-1 protein is highly expressed on the basal cell layer of the epidermis. To examine the possible regulatory mechanism of involucrin gene expression by TEF-1 protein, we analysed involucrin promoter activity of the INV-CAT vector, which was constructed by connecting the 5' upstream region of the involucrin gene (-801 bp upstream from the transcription start site and downstream including the untranslated first exon) to the chloramphenicol acetyltransferase (CAT) reporter gene. The INV-CAT vector was transfected to SV40-transformed human keratinocytes (SVHK). Cotransfection of the TEF-1 expression vector significantly repressed INV-CAT promoter activity in a dose-dependent manner. The repression was also observed by transfection of the GAL4-TEF-1 vector, which was constructed by replacement of the TEF-1 DNA binding domain by the GAL4 activator domain. This suggests that TEF-1-induced repression is due to interference/squelching of a limiting transcriptional intermediary factor that is essential for involucrin expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth Inhibition of SV40-Transformed Human Keratinocytes by TGF-βs Is Not Linked to Dephosphorylation of the Rb Gene Product

We found that transforming growth factor β1 (TGF-β1) and TGF-β2 inhibited the growth of normal human keratinocytes and their SV40-transformed counterpart in a dose dependent manner. Both normal and SV-40 transformed keratinocytes accumulated in G1 when treated with TGF-βs. The hyperphosphorylated form of Rb gene product (RB) was reduced by TGF-βs in normal keratinocytes. In contrast, phosphorylation of RB was not essentially affected in SV-40 transformed cells. This uncoupling of growth kinetics and phosphorylation states of RB in SV-40 transformed keratinocytes suggests that RB is not the primary target of action for TGF-βs and that additional factors or pathways may be involved in mechanisms of the growth inhibition.

The involvement of protein kinase C in proliferation and differentiation of human keratinocytes--an investigation using inhibitors of protein kinase C.

Protein kinase C, the major cellular receptor for tumour-promoting phorbol esters, has been suggested as playing a key role in the regulation of proliferation and differentiation of epidermal cells. In the present study, we investigated the effects of various well-characterized inhibitors of protein kinase C on proliferation and differentiation of SV 40-transformed and normal human keratinocytes. The drugs were found to inhibit cell proliferation in a dose-dependent manner, displaying similar effects in both cell types and reflecting their potencies in inhibiting purified protein kinase C. In contrast, keratinocyte differentiation induced by treatment with a calcium ionophore or spontaneously, i.e. by exposure of cells grown in the presence of low calcium concentration (0.06 mM) to normal calcium concentration (1.6 mM), was not inhibited by the compounds tested. The potent protein kinase C inhibitor, staurosporine, was found even to enhance cell differentiation. Therefore, the present study provides evidence that the classical protein kinase C pathway plays a critical role in the regulation of keratinocyte proliferation rather than in calcium-induced differentiation.

UVB radiation induces phosphorylation of the epidermal growth factor receptor, decreases EGF binding and blocks EGF induction of ornithine decarboxylase gene expression in SV40‐transformed human keratinocytes

We previously demonstrated that epidermal growth factor (EGF) induces a several-fold increase in ornithine decarboxylase (ODC) activity and the steady-state level of ODC mRNA in cultured SV40-transformed human keratinocytes (1). Pretreatment of cell cultures with ultraviolet B (UVB) radiation resulted in a reduction of EGF-induced ODC activity. To determine whether UVB inhibits the accumulation of ODC mRNA by EGF, cells were pretreated with 20 mJ/cm2 UVB or sham-irradiated and then incubated with 100 ng/ml EGF. Northern blot analysis revealed that UVB irradiation entirely blocked the EGF induction of ODC mRNA. Since the binding of EGF to its plasma membrane receptor is the first step in initiating a biological response, the effect of UVB on EGF binding was evaluated. UVB treatment of cultured keratinocytes resulted in an immediate and dose-dependent reduction of EGF binding. Scatchard analysis revealed that the reduction of EGF binding was due to a 52% decrease in the number of available receptors, from 6.2 x 10(4)/cell to 3.0 x 10(4)/cell. However, UVB decreased the EGF-binding affinity very little (Kd = 0.60 nM in control and Kd = 0.75 nM in UVB-treated Z114 cells). In addition, UVB did not alter the rate of EGF internalization. These data suggest that UVB blocks the signal transduction pathway of EGF that is involved in regulation of ODC gene expression. Immunoblot analysis of extracts from irradiated cells showed that UVB induced tyrosine phosphorylation of EGFR and that the quantity of EGFR protein was unaffected by UVB treatment. Phosphorylation of EGFR may be responsible for decreased binding of EGF to its receptor.

Keratin Synthesis in SV40-Transformed Human Keratinocytes after Transient Supertransfection with Harvey Murine Sarcoma Viral DNA

A transformed cell line (HE-IS) was established by transient supertransfection of a SV40-transformed human epidermal cell culture (HE-SL) (R. T. Su and Y.-C. Chang, 1989, Exp. Cell Res. 180, 117-133) with a recombinant plasmid containing an entire Harvey murine sarcoma virus DNA. HE-IS cells showed morphological alterations and less stringent growth requirements in a defined medium. A variant line (HE-TC), selected later from the HE-IS cells after a prolonged maintenance in serum-containing Dulbecco's minimal essential medium (DMEM), was found to be able to grow in either defined or serum-containing medium. Neither HE-IS nor HE-TC cells were able to form colonies in soft agar or induce tumors in athymic mice when inoculated subcutaneously. When grown in defined medium, all transformed cells (HE-SL, HE-IS, HE-TC) revealed a similar keratin pattern as analyzed by two-dimensional gel electrophoresis. However, significant reduction of keratins (notably K5) was detected in HE-TC cells maintained in serum-containing DMEM. The keratin pattern appeared to be regulated by the growth environment. Because of their nontumorigenic characteristics and defined growth properties, these transformed keratinocytes are likely to provide a suitable model system for examining the regulation of cytoskeleton protein synthesis.

Epidermal cell‐shape regulation and subpopulation kinetics during butyrate‐induced terminal maturation of normal and SV40‐transformed human keratinocytes: Epithelial models of differentiation therapy

Recent data indicate that malignant human epidermal cells may be appropriate targets for sodium butyrate (NaB)-mediated differentiation therapy. The response of pre- and post-crisis populations of SV40-transformed human keratinocytes (SVKs) to this differentiation-inducing agent was assessed, therefore, within the framework of NaB-directed normal human keratinocyte (NHK) maturation. NaB augmented cornified envelope (CE) production in NHK and pre-crisis SVK cultures; the time-course and efficiency of induced maturation were similar in the 2 cell systems. In NHKs, the percentage of amplifying ("B" substate) cells decreased with time in NaB correlating with increases in both "C" stage keratinocytes and CEs. The latter formed over one or 2 layers of nucleated basal-like cells. Inductions were accompanied by immediate cell cycle blocks (in both the G1 and G2/M phases), reorganization within the actin cytoskeleton, and transient early increases in cellular actin content. Increased NHK and pre-crisis SVK cytoskeletal-associated actin reached a maximum approximately 48 hr after NaB addition and preceded development of CEs. The CE precursors, thus, probably reside in the "B" substate. Post-crisis SVKs, in contrast, were refractive to NaB-induced terminal maturation or cell-cycle perturbation, failed to initiate actin filament rearrangements, and retained a basal cell-like phenotype. Stable transformation of human SVKs in post-crisis phase, therefore, appears to be associated with loss of maturation "competence" within the "B" keratinocyte subpopulation.