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S M Fischer

Publications and source records attributed to S M Fischer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Coordinated changes in cell cycle machinery occur during keratinocyte terminal differentiation.

Differentiation of cells is typically marked by a cessation of proliferation with a concurrent entrance into a distinct metabolic state marked by tissue specific gene expression. The mechanism by which the cell exits the cell cycle in this process is poorly understood. To determine the potential roles of the cell cycle machinery in the regulation of the terminal differentiation process of epidermal cells, we selected a well characterized in vitro model in which primary mouse keratinocytes are induced to differentiate in response to a raised calcium ion concentration in the medium. The withdrawal from the cell cycle correlates very well with a number of changes in the cell cycle machinery. Changes in the phosphorylation status of the Rb family of proteins occurs coordinately with an increased association of p21, p27 and p57 with cdk2. Furthermore, we find that inhibition of cdk2 activity is not sufficient to elicit changes that occur during keratinocyte differentiation. Finally, the previously described v-Ha-ras block of keratinocyte differentiation correlates with altered regulation of both cyclin D1 and cdk2 suggesting that these genes may play a role in the Ha-ras transformation of a keratinocyte.

Animals↗
PubMed1999

Altered expression of interleukin-1 receptor antagonist in different stages of mouse skin carcinogenesis.

Interleukin-1 receptor antagonist (IL-1Ra) is an endogenous inhibitor of interleukin-1. The expression of IL-1Ra and interleukin-1alpha (IL-1alpha) was measured in murine epidermis after treatment with tumor promoters and in tumor cell lines. A single treatment with three different tumor promoters (12-O-tetradecanoylphorbol-13-acetate (TPA), anthralin, and thapsigargin) induced IL-1Ra mRNA with different kinetics in mouse skin. The expression of IL-1Ra mRNA also was induced by TPA and IL-1alpha in a dose-related and time-dependent manner in cultured mouse keratinocytes. Expression of IL-1Ra mRNA peaked 6 h after treatment. Both IL-1Ra and IL-1alpha protein and IL-1Ra and IL-1alpha mRNA were measured in various keratinocyte tumor cell lines (C50, MT1/2, HEL30, JWF2, CH72, and BPCC2). The expression of IL-1alpha was increased in papilloma and squamous cell carcinoma cell lines. IL-1Ra protein also was increased in nontumorigenic and papilloma cell lines; however, the expression was dramatically reduced in some carcinoma cell lines. Finally, we detected IL-1alpha and IL-1Ra protein in mouse skin tumors by western blot analysis, and localization was assessed by immunohistochemical analysis. Positive staining for both IL-1alpha and IL-1Ra was observed in the cytoplasm and was most prominent in the suprabasal layer. Although IL-1Ra protein increased in papillomas and carcinomas, IL-1alpha protein was not significantly increased above basal level in most tumors.

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

Chemopreventive activity of celecoxib, a specific cyclooxygenase-2 inhibitor, and indomethacin against ultraviolet light-induced skin carcinogenesis.

Epidemiological and dietary studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) reduce the risk of colon cancer, possibly through a mechanism involving inhibition of cyclooxygenase (COX)-2, which is overexpressed in premalignant adenomatous polyps and colon cancer. Because ultraviolet light (UV) can induce COX-2 and nonspecific NSAIDs can decrease UV-induced skin cancer, we evaluated the ability of two compounds, celecoxib (a specific COX-2 inhibitor) and indomethacin (a nonspecific NSAID), to block UV-induced skin tumor development in SKH:HR-1-hrBr hairless mice. Mice fed 150 or 500 ppm celecoxib showed a dose-dependent reduction (60% and 89%, respectively) in tumor yield. Indomethacin (4ppm) reduced tumor yield by 78%. Although both acute and chronic UV exposure increased cell proliferation and edema, neither compound reduced these parameters. In contrast, UV-induced prostaglandin synthesis in the epidermis was effectively blocked by both compounds. UV-induced increases in COX-2 expression in skin were also not altered in any of the treatment groups. Similarly, tumors that constitutively express high levels of COX-2 displayed no reduction by treatment with celecoxib or indomethacin. The dramatic protective effects of celecoxib suggests that specific COX-2 inhibitors may offer a way to safely reduce the risk of skin cancer in humans.

Animals↗
PubMed1999

Mechanism(s) of activation of secretory phospholipase A(2)s in mouse keratinocytes.

The differential activation of different members of the phospholipase A(2) (PLA(2)) superfamily and their regulation are important as one or more of them regulates the production of eicosanoids and others may contribute to the formation of other lipid mediators. We previously reported the existence of two forms of secretory or sPLA(2) in mouse keratinocytes, namely type I and type II sPLA(2). We show here that mouse keratinocyte sPLA(2)s were potently activated by protease treatment and inhibited by protease inhibitors. We also observed that G protein effectors induced substantial release of oleic acid (OA) from prelabeled mouse keratinocytes. A G(i)/G(0) protein activator significantly enhanced the hydrolysis of OA and this increase was not responsive to either pertussis toxin or cholera toxin treatment. Although there was a significant negative correlation between intracellular cAMP levels and OA hydrolysis, experimentally increasing cAMP with forskolin treatment had no effect on sPLA(2) activity. Arachidonic acid but not its metabolites was also shown to marginally activate keratinocyte sPLA(2) by 1.5-fold. These results lead to the conclusion that mouse keratinocyte sPLA(2)s can be regulated primarily by proteolytic activation and a G protein pathway.

Animals↗
PubMed1998

Transcriptional regulation of cyclooxygenase-2 in mouse skin carcinoma cells. Regulatory role of CCAAT/enhancer-binding proteins in the differential expression of cyclooxygenase-2 in normal and neoplastic tissues.

Many studies have suggested that overexpression of cyclooxygenase-2 (COX-2) contributes to the development of tumors in several tissues. COX-2 expression tends to be up-regulated in various types of tumors and transformed cell lines, and the overexpression of COX-2 is caused by enhanced transcription of the gene. In an attempt to characterize the signaling pathway leading to the overexpression of COX-2 in the mouse skin carcinoma cell line JWF2, we investigated cis- and trans-acting factors required for COX-2 expression and demonstrated a molecular mechanism by which COX-2 is expressed differentially in normal and neoplastic tissues. Two regions of the COX-2 promoter containing an E-box and nuclear factor IL6 site were identified as the positive regulatory elements through transient transfections with luciferase reporter vectors containing the various 5'-flanking regions of the promoter. Moreover, electrophoretic mobility shift assays and cotransfection experiments showed that upstream stimulatory factors and CCAAT/enhancer-binding proteins (C/EBPs) bind to the E-box and nuclear factor IL6 site, respectively, and functionally transactivate the COX-2 promoter. We also found that C/EBP isoforms are expressed differentially during mouse skin carcinogenesis, suggesting that overexpression of COX-2 in tumors may be caused by a change in C/EBP expression levels.

Activating Transcription Factors↗
PubMed1998

Suppression or elevation of cytosolic phospholipase A2 alters keratinocyte prostaglandin synthesis, growth, and apoptosis.

Prostaglandins and other arachidonic acid (AA) metabolites are synthesized by keratinocytes in response to tumor promoters and are produced at very high levels in tumors. After phorbol ester treatment, AA is hydrolyzed from keratinocytes primarily by the cytosolic form of phospholipase A2 (cPLA2), which exhibited a strong substrate preference for phosphatidylcholine over phosphatidylethanolamine and AA over other fatty acids. Phorbol esters increase cPLA2 activity but not the level of expression. To dissociate increased cPLA2 activity from other phorbol ester effects and thus determine the effects of altered AA release on cell growth, the murine keratinocyte cell line, HEL-30, was stably transfected with the sense or antisense cDNA for cPLA2. The resulting cell lines displayed corresponding over- or underexpression and up to 23-fold differences in cPLA2 activity between them. Phorbol ester caused a 15-fold difference in AA release between sense and antisense transfectants. Prostaglandin E2 levels correlated with AA release levels. The sense transfectants showed an enhanced proliferative capacity, based on increased cell number over time and [3H]thymidine incorporation. The antisense transfectants had significantly (>60%) reduced growth rates, compared with both parental cells and sense transfectants. The extent of apoptosis was determined in tumors from cell lines grown in graft chambers in vivo. The number of apoptotic cells was significantly greater in tumors from the sense transfectants, based on terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining, compared with the parental or antisense lines. These data are in agreement with a recent study (M. C. Stern et al., Mol. Carcinog., 20: 137-142, 1997) showing a correlation between increased apoptosis and tumor progression in this model system. These results suggest that the elevated eicosanoid synthesis that is observed in skin carcinomas contributes to the growth and progression of these tumors.

Animals↗
PubMed1998

The effect of vitamin E acetate on ultraviolet-induced mouse skin carcinogenesis.

Despite the benefits of sunscreens, ultraviolet (UV) exposure can still lead to skin cancer. In this study we investigated the effect of topical application of the antioxidant vitamin E acetate (VEA) on the inhibition of UV-induced carcinogenesis. Hairless SKH-1 mice received 5.2 mg of VEA 30 min before (VEA/UV) or after (UV/ VEA) a single minimal erythemic dose of UV light. Vehicle-control animals received acetone 30 min before UV exposure (Ace/UV). After 24 h, cyclobutane dimer repair was twofold and 1.5-fold greater in the UVNEA and VEA/UV groups, respectively. Expression of p53 protein in the UV/VEA group was maximum at 12 h after UV exposure, whereas in the Ace/UV- and VEA/UV-treated mice, maximum p53 immunostaining was statistically higher at 15 h (P = 0.03). DNA synthesis as determined by 5-bromo-2'-deoxyuridine incorporation was twofold higher after 15 h in all groups but was not statistically different among treatment groups. Protein levels of cyclin D1 and p21 were increased in both VEA groups by 6 h. In addition, VEA treatments delayed tumor formation and yield for the first 20 wk, although this difference was lost by 30 wk. The telomerase activity of carcinomas from the UV/VEA-treated mice was statistically lower than that of the Ace/UV-treated mice (P = 0.05). This study showed that although VEA may mitigate some of the initial events associated with UV irradiation such as DNA damage and p53 expression, it has limited potential in preventing UV-induced proliferation and tumor formation.

Animals↗
PubMed1998

Analysis of two inbred strains of mice derived from the SENCAR stock with different susceptibility to skin tumor progression.

The SENCAR stock of mice has proved to be a useful model in dissecting out the multistage nature as well as the critical mechanisms involved in skin tumorigenesis. This outbred stock was selectively bred to be susceptible to initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). In order to obtain mice more suitable for genetic analyses of tumor susceptibility and tissue transplantation studies, several inbred lines of mice were derived from the SENCAR stock. One of these lines, the SSIN mice, has a higher susceptibility to tumor promotion compared to the SENCAR stock but is very resistant to tumor progression. On the other hand, the SENCAR B/Pt mice, derived also from the outbred stock, not only have a tumor promotion susceptibility almost identical to the SSIN mice, but they also have a high susceptibility to tumor progression. In order to understand the nature of the phenotypic differences between these two inbred lines we have characterized them using several parameters and markers that are associated with the progression of papillomas to squamous cell carcinoma (SCC). In this sense we analysed the tumor multiplicity and SCC incidence, and the expression of markers of progression and cell cycle related proteins in papillomas derived from both strains. Our results showed that while both strains have a similar papilloma multiplicity and incidence the SENCAR B/Pt mice have 67% incidence of SCC, compared to 0% in the SSIN. SENCAR B/Pt papillomas at 30 weeks of promotion have a higher and aberrant expression of K13, and loss of connexin 26. TGF-beta1 was found to be over-expressed in the suprabasal and superficial cells in the SENCAR B/Pt papillomas, while it was only expressed in the superficial cell layer in those derived from SSIN. The SENCAR B/Pt papillomas also showed an enlarged proliferative compartment with overexpression of cyclin D1 and PCNA as seen by immunohistochemistry and Western blot.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1998

Temporal distributions of problem behavior based on scatter plot analysis.

The scatter plot is a commonly used assessment tool for identifying temporal patterns in the occurrence of behavior problems. However, the extent to which such patterns are frequently observed is unknown because little research has evaluated the general utility of the scatter plot. We conducted a large-scale analysis of within- and across-day occurrences of problem behavior by conducting continuous observations of 20 individuals living in four residential facilities. Data were recorded during 30-min intervals throughout participants' waking hours for 30 days by direct care staff and were converted into scatter plot formats. Five sets of data were excluded from further analysis due to poor interobserver agreement (below 80%). Visual analysis of the remaining 15 scatter plots indicated that none showed any reliable temporal pattern of responding. However, when the data were transformed into aggregate "control charts" based on statistical process control procedures, 12 of the 15 sets of data revealed one or more 30-min intervals during which problem behavior was more likely to occur. Results are discussed in terms of the practicality of applying statistical analyses to scatter plot data and of collecting data for the length of time needed to show statistical significance. It was concluded that detailed functional or descriptive analyses, which would reveal cause-effect or correlational relationships between behavior and specific environmental events, may be both more precise and more efficient forms of assessment.

Adolescent↗
PubMed1998

Identification and characterization of several forms of phospholipase A2 in mouse epidermal keratinocytes.

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the release of arachidonic acid (AA) from mouse keratinocytes. A distinct difference was observed between the fatty acid release profile elicited by TPA and other stimuli. These findings led to the investigation of keratinocyte phospholipase A2 (PLA2), which catalyzes the release of sn-2 fatty acids from membrane phospholipids and regulates the production of eicosanoids. We characterized and identified several forms of PLA2 in mouse keratinocytes, a cytosolic or cPLA2 and two secretory or sPLA2s in the membrane. The PLA2 in keratinocyte cytosol is sensitive to heating and acid treatment, while resistant to reducing reagent. The PLA2 in keratinocyte membrane is resistant to heating and acid treatment, while sensitive to reducing reagent. These characteristics suggested the presence of a cPLA2 and at least one type of sPLA2. Inhibitor data further confirmed the identities of these PLA2s. The cPLA2 was activated by TPA, and appeared to be responsible for the majority of the specific release of AA observed in mouse keratinocytes treated with TPA. The calcium ionophore A23187, and 4alpha-TPA did not elicit the selectivity towards AA observed with TPA. The release of linoleic acid (LA) and oleic acid (OA) from A23187- and 4alpha-TPA-treated keratinocytes suggests activation of sPLA2. These activities may be due to the existence of both type I and type II sPLA2, as both were identified by polymerase chain reactions. In conclusion, keratinocytes express several forms of phospholipase A2 that differ in their substrate specificities and mechanisms of activation, resulting in distinct agonist-specific fatty acid release profiles.

Animals↗
PubMed1997

Prostaglandins and cancer.

Prostaglandins (PGs) are lipids produced enzymatically and nonenzymatically from 20 carbon fatty acids, particularly arachidonic acid. Prostaglandins and related metabolites (collectively referred to as eicosanoids), including the thromboxanes, prostacyclins, hydroperoxy and hydroxy fatty acids, and leukotrienes are produced by most tissues of the body by oxidation of arachidonic acid, although the amount and class of product varies with cell type. The biological action of many of these eicosanoids as key regulators of cell processes that range from proliferation to adhesion and migration are currently being elucidated. An association of high levels of PGs and their synthetic enzymes, the PG synthases/cyclooxygenases, have been noted for many of the major types of cancer. Prostaglandins may contribute to the cancer processes through one or more of several mechanisms including increased proliferation, apoptosis, enhanced carcinogen metabolism or modulation of the immune system. The recent understanding of the regulation of substrate availability and of the regulation (or dysregulation in many neoplasias) of the synthetic enzymes has opened avenues leading to the design of isozyme specific inhibitors and better cancer prevention strategies. However, the apoptosis caused by these inhibitors as well as other drugs has raised some question concerning the relative importance of PGs. This is an issue that remains to be resolved.

Animals↗
PubMed1997

Uncoordinated regulation of mRNA expression of the three isoforms of transforming growth factor-beta in the mouse skin carcinogenesis model.

The mRNA expression and autoregulation of expression of the three isoforms of transforming growth factor-beta (TGFbeta) were examined in the mouse skin carcinogenesis model by northern analyses. We found that TGFbeta3 mRNA levels followed a pattern similar to those of TGFbeta1 during carcinogenesis: the levels were somewhat low in normal skin but became highly overexpressed in late-stage papillomas and squamous cell carcinomas (15- to 20-fold higher than in normal skin). On the other hand, the TGFbeta2 mRNA levels remained relatively low in all benign and malignant tumors, even though the levels were higher than the nearly undetectable levels in normal skin. In a squamous cell carcinoma cell line (CH72), stable transfection and expression of a mutated simian TGFbeta1 cDNA producing bioactive TGFbeta1 significantly downregulated (mean greater than ten-fold) TGFbeta2 mRNA levels and modestly downregulated (about twofold) murine TGFbeta1 expression but had no effect on TGFbeta3 mRNA. In contrast, treatment of all CH72 clones with exogenous TGFbeta1, TGFbeta2, or TGFbeta3 either had no effect or slightly downregulated TGFbeta1 mRNA, upregulated TGFbeta2 mRNA expression an average of twofold to threefold, and strongly upregulated (mean 13- to 27-fold) TGFbeta3 mRNA levels. TGFbeta treatment of primary cultures of mouse skin keratinocytes upregulated all three TGFbeta mRNA levels slightly to moderately (1.3- to 5-fold). Thus, although TGFbeta1 and TGFbeta3 mRNA expressions were apparently coordinately upregulated during mouse skin carcinogenesis, the three TGFbeta mRNAs were differentially regulated by stable transfection of active TGFbeta1 versus exogenous TGFbeta treatment in CH72 cells and by TGFbeta treatments of normal keratinocytes versus carcinoma CH72 cells.

Animals↗
PubMed1997

Opposite effect of stable transfection of bioactive transforming growth factor-beta 1 (TGF beta 1) versus exogenous TGF beta 1 treatment on expression of 92-kDa type IV collagenase in mouse skin squamous cell carcinoma CH72 cells.

We have previously shown that transforming growth factor-beta 1 (TGF beta 1) mRNA is consistently overexpressed in squamous cell carcinomas relative to normal mouse skin. Here we show that 92-kDa type IV collagenase (matrix metalloproteinase) (MMP-9) mRNA was likewise progressively overexpressed during mouse skin carcinogenesis. To determine if overexpression of MMP-9 and TGF beta 1 are linked, we stably transfected a bioactive TGF beta 1 into a mouse skin squamous cell carcinoma cell line (CH72), which resulted in about twofold to three-fold higher levels of secreted active TGF beta 1. Active TGF beta 1-transfected cells grew only slightly, but not significantly, more slowly in vitro and in vivo than vector-only transfectants. Two clones overexpressing active TGF beta 1 secreted much reduced levels of MMP-9 activity, as determined by zymogram analyses. However, treatment of these clones with 40 pM exogenous TGF beta 1 for 48 h enhanced secretion of MMP-9 activity. Constitutive mRNA expression of MMP-9 was reduced twofold to 70-fold in five untreated active TGF beta 1-transfected clones relative to the other transfectants. In contrast, treatment with 40 pM exogenous TGF beta 1 induced MMP-9 mRNA expression in a time-dependent fashion, from twofold to fourfold after 4 h to a maximum of 12- to 19-fold after 24-48 h. Induction of MMP-9 mRNA was dose dependent at TGF beta 1 concentrations of 4-400 pM. Thus, stable transfection of bioactive TGF beta 1 downregulated whereas exogenous TGF beta 1 treatment upregulated MMP-9 activity and expression. Treatment of transfectants with a neutralizing TGF beta 1 antibody slightly downregulated constitutive MMP-9 mRNA (20-30%) but completely blocked induction by exogenous TGF beta 1. Thus, the effect of TGF beta 1 transfection was not due to secreted TGF beta 1 but may have been a secondary effect.

Animals↗
PubMed1997

Identification of an autocrine mechanism for regulating cell-cycle progression in murine keratinocytes.

Primary murine keratinocytes possess a limited doubling potential regardless of plating density or the inclusion of competence factors insulin, epidermal growth factor, and/or fetal bovine serum within the culture medium. In contrast, a murine cell line (CH-72), derived from a 7,12-dimethylbenz[a]-anthraceneinitiated, 12-O-tetra-decanoylphorbol-13-acetate-promoted mouse skin carcinoma, was found to exhibit unlimited proliferative potential; this was demonstrated by the ability of these cells to produce the progression factor required for entry into the DNA-synthesis phase in the absence of competence-factor stimulation. Conditioned medium, collected from murine carcinoma cells, was subsequently shown to increase the level of [3H]thymidine incorporation in competence-factor-deprived CH-72 cultures by more than a factor of 4 within 16 h. Moreover, consistent with its ability of recruit cells cycling within the first gap phase directly into the DNA-synthesis phase, the autocrine progression factor present in conditioned medium decreased the G1:S ratio from the 55:29 observed with growth medium controls to 38:46. Preliminary characterization of the autocrine factor produced by cultured murine carcinoma cells using gel-filtration chromatography revealed a molecular mass of less than 2 kDa, similar in size to the factor previously shown by our laboratory to promote G1-phase progression in cultures of normal human foreskin keratinocytes.

Animals↗
PubMed1997

Epidermal proliferation but not quantity of DNA photodamage is correlated with UV-induced mouse skin carcinogenesis.

The hairless SKH-1 mouse strain has a higher skin tumor incidence, shorter tumor latency, and higher tumor yield in response to ultraviolet (UV) irradiation than the SENCAR strain. In this study we assessed the differences in UV susceptibility of both strains by measuring DNA photodamage and epidermal proliferation after one UV treatment and after 1, 3, 6, and 9 wk of chronic UV irradiation. Induction rates for cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4) PDs] were significantly greater in the SKH-1 strain than the SENCAR strain, but no strain differences in repair kinetics were detected for CPDs or (6-4) PDs. With chronic UV exposure we observed the following: (i) there was an equal amount of DNA photodamage in both strains; (ii) the number of (6-4) PDs was significantly greater than the CPDs after 6 wk; (iii) there were a significantly greater number of epidermal cells (1.5-fold increase) in the SKH-1 strain; (iv) the number of cycling cells, as measured by 5-bromo-2'-deoxyuridine (BrdU), were located both basally and suprabasally and were significantly greater in the SKH-1 strain; and (v) the number of cells immunoreactive to p53 was equivalent in both strains, but immunoreactive cells were located suprabasally in the SKH-1 strain after 9 wk of UV. These results show that the etiologic role of UV in tumorigenesis is dependent on events other than the amount of DNA photodamage in mouse epidermis.

Animals↗
PubMed1997

Noncontingent delivery of arbitrary reinforcers as treatment for self-injurious behavior.

Results of recent research have shown that noncontingent reinforcement (NCR) can be effective in reducing the frequency of behavior problems. In typical NCR applications, the reinforcer that is responsible for behavioral maintenance (as demonstrated through a functional analysis) no longer follows occurrences of the target behavior but instead is delivered according to a time-based schedule. Thus, it is unclear if NCR would be effective if the target behavior continued to be reinforced or if arbitrary reinforcers (i.e., those irrelevant to behavioral maintenance) were substituted for the maintaining reinforcers in the NCR procedure. In this study, 2 individuals whose self-injurious behavior (SIB) was maintained by positive reinforcement were exposed to conditions in which arbitrary and maintaining reinforcers were withheld and were delivered either contingently or noncontingently. Results indicated that noncontingent delivery of arbitrary reinforcers was effective in reducing SIB even though occurrences of SIB produced access to the maintaining reinforcer. These results suggest that (a) arbitrary reinforcers may sometimes be substituted for maintaining reinforcers, (b) an important component of NCR procedures is alteration of a behavior's establishing operation, and (c) NCR with arbitrary reinforcers might therefore be effective when maintaining reinforcers cannot be identified or withheld during the course of treatment.

Adult↗