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H Hennings

Publications and source records attributed to H Hennings.

At least 37 records · Page 2Linked to original sources
PubMed1992

Development of an in vitro model to study carcinogen-induced neoplastic progression of initiated mouse epidermal cells.

Initiation and promotion in mouse skin carcinogenesis produce multiple benign tumors, squamous papillomas, but only a few squamous cell carcinomas. The spontaneous conversion from the benign to the malignant phenotype occurs over many months and in stages, but induced malignant conversion can be accomplished more rapidly by exposure of papilloma-bearing mice to mutagens or by transfection of papilloma cell lines with specific oncogenes. The analysis of genetic targets responsible for carcinogen-induced neoplastic progression would be facilitated by the development of in vitro models where the process is rapid, focal, and quantitative. To this end, primary newborn mouse keratinocytes were initiated in vitro by the introduction of the v-rasHa oncogene via a defective retrovirus. Recipient cells produce squamous papillomas and have a high proliferation rate in culture medium with 0.05 mM Ca2+, but fail to grow in medium with 0.5 mM Ca2+ which is permissive for growth of malignant keratinocytes. When v-rasHa-keratinocytes were exposed to mutagens in vitro, proliferative foci emerged after culture in 0.5 mM Ca2+ for 4 weeks. These foci stained intensely red with rhodamine stain, could be easily quantitated, and readily incorporated bromodeoxyuridine. Dose-response studies with several mutagens indicated that the number of foci increased with concentration to the point where excessive cytotoxicity developed. Mutagens varied in potency for producing foci in the following order: cis-diamminedichloroplatinum greater than or equal to benzo(a)pyrene diolexpoxide I greater than N-methyl-N'-nitro-N-nitrosoguanidine greater than or equal to 4-nitroquinoline-N-oxide greater than N-acetoxy-acetyl- aminofluorene. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate was inactive in the assay. A subset of cell lines derived from foci produced malignant tumors in vivo, while others were not tumorigenic. Analysis of DNA from cell lines and tumors revealed that most tumorigenic cell lines maintained the v-rasHa genome, whereas the viral sequences were deleted in nontumorigenic cell lines. Immunohistochemical analysis indicated that proliferative foci and quiescent v-rasHa keratinocytes expressed keratin 8, a marker of v-rasHa expression in cultured keratinocytes. Cells in foci, but not v-rasHa control cells, expressed keratin 13, a marker which is strongly associated with the malignant progression of skin tumors in vivo. This in vitro assay provides a quantitative model to study chemically induced focal neoplastic progression at the cellular level and to identify agents which may be selective for enhancing malignant conversion.

Animals↗
PubMed1992

A model for initiated mouse skin: suppression of papilloma but not carcinoma formation by normal epidermal cells in grafts on athymic nude mice.

The availability of a skin grafting system on nude mouse hosts and of epidermal cell lines which form papillomas when grafted has made possible the creation of a model for initiated skin in vivo from cultured cells. When grafted with 6-8 x 10(6) primary dermal fibroblasts, 10 x 10(6) primary epidermal cells form an apparently normal skin, and cell line SP-1 (0.5 x 10(6) cells) forms papillomas. Cell line SP-1 was derived from papillomas produced on SENCAR mice by initiation with 7,12-dimethylbenzaadaa]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate. Grafting of 0.5 x 10(6) SP-1 cells along with 10 x 10(6) SENCAR newborn primary epidermal cells resulted in a 90% reduction in the average papilloma volume per mouse compared to controls without primary epidermal cells. Suppression occurred specifically with epidermal cells, either cultured or freshly prepared, and was not seen when an equivalent number of SENCAR primary dermal fibroblasts was grafted in place of epidermal cells. Nor did suppression occur when primary epidermal cells were replaced with a carcinogen-altered cell line, SCR722. SCR722 cells have a normal-skin phenotype when grafted. Furthermore, suppression of tumor formation did not occur when a malignant variant of SP-1 cells replaced benign SP-1 cells in grafts. Repeated treatment of suppressed grafts with 12-O-tetradecanoylphorbol-13-acetate resulted in an increased number of mice with papillomas and a larger mean papilloma volume per mouse compared to controls treated with solvent alone, whereas treatment of nonsuppressed grafts of papilloma cells with promoter produced no change in tumor size. These results support the concepts that normal epidermal cells suppress the growth of initiated cells and that repeated treatment with phorbol ester tumor promoters overcomes the suppression, leading to benign tumor formation.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

Activity of diverse tumor promoters in a keratinocyte co-culture model of initiated epidermis.

The suppression of focal growth of initiated mouse keratinocytes by co-culture with normal keratinocytes can be overcome by treatment of co-cultures with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). This keratinocyte co-culture model was developed as an analog of initiated mouse epidermis to facilitate the study of tumor promotion in cell culture. A number of promoters of TPA-type [those with protein kinase C (PKC) as receptor] were compared to non-TPA-type promoters for activity in the keratinocyte co-culture model. The TPA-type promoters teleocidin and aplysiatoxin show comparable activity to that of TPA. Exposure of co-cultures to oleoyl-2-acetylglycerol, a diacylglycerol activator of PKC, also induces focal outgrowth of initiated cells, suggesting that PKC is likely to be involved in the mechanism of action of these compounds. However, the involvement of alternative pathways (not involving PKC directly) for clonal selection are evident since the non-TPA-type promoters okadaic acid, staurosporine and thapsigargin are also active in the assay. Thus, the keratinocyte co-culture model differs from fibroblast models of normal and neoplastic co-cultures in which only TPA-type promoters are active. In further contrast to certain fibroblast co-cultures, TPA does not inhibit cell-cell communication under conditions that suppress focus formation. Taken together with previous data, we conclude that the keratinocyte co-culture model may have broad application for detecting skin tumor promoters, and may be useful for dissecting the mechanism by which normal epidermal cells suppress the growth of initiated cells.

Animals↗
PubMed1991

Differences in the regulation of intracellular calcium in normal and neoplastic keratinocytes are not caused by ras gene mutations.

The development of resistance to terminal differentiation is an early event in epidermal neoplasia. Altered differentiation can be detected in vitro since normal epidermal cells are induced to differentiate in medium with Ca2+ greater than 0.1 mM while neoplastic epidermal cells and keratinocytes transduced with a v-rasHa gene are resistant to Ca2+. In normal epidermal cells, the elevation of extracellular Ca2+ (Cao) from 0.05 to 1.2 mM causes a biphasic intracellular Ca2+ (Cai) response in which a transient (10 min) peak of 4-5-fold over basal values is followed by a sustained (greater than 24 h) 2-fold increase in steady-state Cai. The transient peak in Cai is dependent on a serum component and independent of Cao, while the sustained plateau is directly dependent on Cao. The transient peak responding to a serum factor is lost in normal cells after 24 h in 1.2 mM Ca2+, a time when these cells are differentiating. Two neoplastic keratinocyte cell lines, SP-1 and 308, which produce benign tumors in vivo, also have a biphasic Cai response to an increase in Cao. In these cells, the transient peak is also serum dependent and amplified to 10-fold over basal values. However, the plateau value is not sustained and returns to basal values by 8 h, independent of Cao. Furthermore, 308 cells remain sensitive to the serum-induced Cai transient after 24 h in 1.2 mM Ca2+. To determine whether the activating c-rasHa mutation in 308 and SP-1 cells was responsible for the altered Cai regulation, a v-rasHa gene was introduced into normal keratinocytes by a defective retrovirus. This also produces the papilloma phenotype in vivo. Recipient cells were resistant to Ca(2+)-induced terminal differentiation although they did not proliferate in 1.2 mM Ca2+. The Cai profile in response to 1.2 mM Ca2+ was identical in normal and v-rasHa keratinocytes, and these cells lost the serum-induced transient Cai peak after 24 h. Thus, the activation of the c-rasHa gene in 308 or SP-1 cells is probably not solely responsible for the altered Cai response in neoplastic cell lines. Sustained physiological elevation of Cai may be relevant to the loss of proliferative potential in both normal and v-rasHa keratinocytes in 1.2 mM Ca2+. In addition, v-rasHa-mediated or activated c-rasHa-mediated changes in a complementary pathway may contribute to the block in terminal differentiation in neoplastic cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Malignant conversion, the first stage in progression, is distinct from phorbol ester promotion in mouse skin.

Papillomas induced by DMBA initiation-TPA promotion protocols are necessary precursor lesions of squamous cell carcinomas. The papillomas are heterogeneous in their potential for progression to carcinomas, a property apparently induced at the time of initiation. The probability of conversion to malignancy is highest for the papillomas most easily promoted, by either the first few TPA treatments or by "weak" promoters such as mezerein or chrysarobin. The conversion frequency is lowest for TPA-dependent papillomas and those papillomas which appear late in a TPA promotion protocol. The spontaneous rate of malignant conversion is not altered by continued TPA treatment; TPA promotion may simply expand clones of initiated cells which are already programmed with a given probability of conversion. Treatment of papilloma-bearing mice with a genotoxic agent, such as 4-NQO, urethane or cisplatin, increases the rate of malignant conversion. The properties of active converting agents differ markedly from those of the phorbol ester promoting agents, suggesting differences in the mechanisms of action of these two classes of compounds. A genetic mechanism appears likely to explain conversion. The differences between the two stages are further emphasized by the finding that inhibitors of tumor promotion are not inhibitory when given during malignant conversion. The converting agent urethane also affects the subsequent discrete stage in tumor progression, tumor metastasis. Differences in metastatic potential have been found between carcinomas which progress spontaneously after TPA promotion and carcinomas induced in TPA-promoted papillomas by urethane. The multistage nature of experimental epidermal carcinogenesis is well established. The mouse skin model will continue to be valuable for mechanistic studies since similar stages have been described in other tissues as well as in man.

4-Nitroquinoline-1-oxide↗
PubMed1991

Regulation of intracellular free calcium in normal murine keratinocytes.

Cultured normal murine keratinocytes maintain a basal cell phenotype in medium with a Ca2+ concentration of 0.05 mM and differentiate when exposed for 28-48 h to medium supplemented with extracellular Ca2+ greater than 0.10 mM. Previous studies have documented Ca2+ activation of signaling pathways in the plasma membrane and tightly regulated cellular responses to small incremental changes in extracellular Ca2+. To determine if changes in free cytosolic calcium (Cai) are associated with these early signaling events, digital image analysis of fura-2-loaded keratinocytes was used to measure Cai in individual cells. Basal keratinocytes in 0.05 mM Ca2+ display a biphasic Cai increase when exposed to greater than 0.1 mM Ca2+ in serum-containing medium. These separate phases were controlled by different media components. Initial peak Cai occurred rapidly (within 60 s), was transient (lasting less than 5 min), and resulted from release of 10-20% of total intracellular Ca2+ stores. Peak Cai depended on serum concentration and was independent of extracellular Ca2+. This transient Cai response was lost as keratinocytes differentiated. Plateau Cai level was sustained (greater than 24 h) and depended on extracellular Ca2+, but not serum. The magnitude of plateau Cai increased incrementally following increases in extracellular Ca2+ as small as 0.02 mM. A similar biphasic Cai increase was noted in cultures of murine dermal fibroblasts stimulated by 1.2 mM Ca2+ and serum. However, fibroblasts did not lose the serum response in high-Ca2+ medium, and plateau Cai was not sensitive to small changes in extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Coculture of neoplastic and normal keratinocytes as a model to study tumor promotion.

In order to study skin tumor promotion, a cell culture model system analogous to initiated mouse epidermis was developed. Keratinocytes of the neoplastic cell line 308 display the initiated phenotype since papillomas are produced when the cells are grafted to the backs of athymic mice. Coculture of a small number of these initiated cells with confluent normal keratinocytes results in the suppression of growth of colonies of 308 cells. This inhibition, which is calcium dependent, epidermal cell specific, and requires cell contact, can be overcome by exposure to various tumor promoters. Promoters and antipromoters of diverse structure and mechanism of action are recognized in this keratinocyte coculture model.

Animals↗
PubMed1991

Alterations in epidermal biochemistry as a consequence of stage-specific genetic changes in skin carcinogenesis.

The induction of cancer on mouse skin by initiation-promotion protocols occurs through stages in which a benign squamous papilloma is an obligate precursor of squamous cell carcinoma. Activation of the Ha-ras gene is sufficient to produce the papilloma phenotype, while additional genetic changes are required for malignant conversion. The introduction of Ha-ras into normal keratinocytes suppresses the expression of differentiation markers, keratin K1 and K10, and loricrin (a cornified envelope precursor) and, to a lesser extent, filaggrin, at the level of transcription. However, cells initiated by Ha-ras express a nonepidermal keratin, K8. The transcription of K8 in these cells is sensitive to the level of medium Ca2+, being abundant in 0.5 mM Ca2+ and not detected in 0.05 mM Ca2+. Epidermal differentiation is regulated by signalling, which involves changes in phosphatidylinositol turnover and intracellular Ca2+. Cells initiated by Ha-ras do not differ from normal keratinocytes in their intracellular Ca2+ response patterns, at least in response to changes in extracellular Ca2+ and serum factors. However, c-Ha-ra keratinocytes have a high basal level of phosphatidylinositol (PI) turnover, which is additive with several other inducers of this pathway, including Ca2+ and aluminum fluoride. Additional studies suggest that high turnover of the PI pathway is incompatible with differentiation-specific gene expression in keratinocytes. We suggest this negative relationship is mediated through elevated diacylglycerol production and chronic down-modulation of protein kinase C. Protein kinase C is known to be essential for expression of differentiation-related genes in keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Activation of the rasHa gene in urethane-initiated papillomas induced by protocols with high and low frequencies of malignant conversion.

The number of papillomas that develop in mice initiated with a single exposure to urethane and promoted by repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) is increased greater than 4-fold by pretreating the skin once with TPA 24 hr before administration of urethane. In contrast, the carcinoma incidence was increased only 2-fold by the TPA pretreatment. Individual papillomas developed from the two protocols, differing in the potential for conversion to malignancy, were compared for activation of the rasHa gene. An activated oncogene that transformed 3T3 cells was found in the DNA of four of five papillomas from urethane-initiated, TPA-promoted mice and in eight of eleven papillomas from similarly promoted mice exposed to TPA before urethane initiation. Southern analysis of DNA from tumors or 3T3 foci demonstrated that the rasHa gene was activated by an A----T transversion at the second base of codon 61 in all mutated alleles. Thus, tumors induced by the two protocols did not differ in rasHa activation.

3T3 Cells↗
PubMed1990

Development of an in vitro analogue of initiated mouse epidermis to study tumor promoters and antipromoters.

To facilitate the study of skin tumor promotion, a cell culture model system with characteristics analogous to initiated mouse epidermis was established. Cells of the keratinocyte cell line 308, derived from adult mouse skin initiated with 7,12-dimethylbenz[a]anthracene, display the initiated phenotype, since papillomas are produced when the cells are grafted to the backs of athymic mice. Coculture of a small number of these initiated cells with confluent normal primary keratinocytes resulted in the inhibition of growth of colonies of 308 cells. Addition of fresh keratinocytes weekly was required to sustain the inhibition for 3-4 weeks. Inhibition of 308 cell colonies required culture medium with a calcium concentration of 1.2 mM; normal keratinocytes did not inhibit 308 cells in medium with 0.05 mM calcium. Growth of 308 cells was not inhibited by coculture with confluent fibroblasts or by 1.2 mM calcium medium conditioned by either keratinocytes or fibroblasts. During continuous exposure of the cocultures to tumor promoters, 308 cell colonies became apparent within 2-3 weeks. A limited number of promoters were tested in this model system and 12-O-tetradecanoylphorbol-13-acetate, 12-O-retinoylphorbol-13-acetate, mezerein, and benzoyl peroxide were all active. The number of colonies which developed during promoter exposure in cocultures showed a dose-response curve which differed from the dose-response curve for stimulation of growth of 308 colonies in the absence of normal keratinocytes. Simultaneous treatment with 12-O-tetradecanoylphorbol-13-acetate and known inhibitors of skin tumor promotion, such as retinoic acid, fluocinolone acetonide, and bryostatin 1, blocked colony formation of 308 cells in cocultures but not in cultures with only 308 cells. In this model system, the actions of promoters and inhibitors both appear to be mediated by normal keratinocytes.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Enhanced malignant conversion of benign mouse skin tumors by cisplatin.

The chemotherapeutic agent cisplatin, reported to be a complete carcinogen in rodents and a tumor initiator for mouse skin, was tested for activity to enhance the conversion of carcinogen-induced skin papillomas to carcinomas. Initiation of mouse skin by 7,12-dimethylbenz[a]anthracene followed by 12 weeks of promotion by 12-O-tetradecanoylphorbol-13-acetate produced seven to eight papillomas/mouse. Ten weekly injections of 100 micrograms of cisplatin into these papilloma-bearing mice induced a 2.3-fold enhancement of conversion relative to the spontaneous rate of 1.9%. Even a single exposure to cisplatin in tumor-bearing mice increased the carcinoma incidence to the same extent as 10 exposures to urethane, an agent previously shown to enhance malignant conversion. At the dose tested, cisplatin was inactive as a complete carcinogen or a tumor promoter. Cisplatin-DNA adducts, measured in samples from skin, liver, and kidneys, were persistent for at least 4 weeks after the last exposure to cisplatin. Thus cisplatin is a relatively potent inducer of the putative genotoxic changes required for conversion of skin tumors from a benign to a malignant phenotype. The activity of cisplatin in the initiation and malignant conversion stages in this animal model for carcinogenesis suggests that patients given cisplatin-based chemotherapy are at increased risk for the development of treatment-induced second cancers.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Sensitivity of subpopulations of mouse skin papillomas to malignant conversion by urethane or 4-nitroquinoline N-oxide.

Papillomas induced in SENCAR mice by initiation with 7,12-dimethylbenz(a)anthracene and promotion by treatment for 10-12 weeks with 12-O-tetradecanoylphorbol-13-acetate (TPA) convert to malignancy at a low frequency. The rate of malignant conversion can be increased by either (a) promoting with TPA for a shorter duration or (b) treatment of papilloma-bearing mice with certain genotoxic chemicals, such as 4-nitroquinoline N-oxide (4-NQO) or urethane. The spontaneous conversion rate of papillomas promoted by 5 weeks of TPA exposure is severalfold higher than that of papillomas arising later during TPA promotion. Here, we compared the sensitivity to the converting agents 4-NQO and urethane of papillomas promoted by TPA for either 5, 10, or 20 weeks. In the mice promoted for 5 weeks with TPA, the already high spontaneous conversion frequency was increased 2.5 times by 4-NQO. A 2-fold increase was found after 10 weeks of TPA promotion. In contrast, no increase was seen with 4-NQO exposure begun after 20 weeks of TPA promotion. Similar results were found with urethane as converting agent. The sensitivity of the papillomas induced by short-term TPA treatment to induced conversion remains high even after a 16-week period without TPA treatment; when urethane exposure was delayed until week 21 after TPA promotion for weeks 1-5, a 2.4-fold increase in the conversion frequency was observed.

4-Nitroquinoline-1-oxide↗
PubMed1990

The malignant conversion step of mouse skin carcinogenesis.

Multiple benign squamous papillomas commonly precede the development of an occasional squamous cell carcinoma in mouse skin carcinogenesis. The incidence of carcinomas can be enhanced by treating papilloma-bearing mice with mutagens such as urethane, nitroquinoline-N-oxide, or cisplatinum. This observation suggests that a genetic change is required for malignant conversion. The malignant phenotype is characterized by a marked reduction in the transcription of specific epidermal differentiation markers, a pattern which is useful for the early diagnosis of malignant conversion. Cells expressing a benign phenotype can be obtained by introducing the v-rasHa oncogene into cultured epidermal cells by a replication-defective retrovirus. Alternatively, benign tumor cells can be cultured from papillomas induced by chemical carcinogens in vivo or from carcinogen-treated mouse epidermis. In all cases, the benign phenotype in vitro is characterized by an altered biological response to changes in extracellular calcium, an important determinant of the differentiation state of cultured normal keratinocytes. Transfection of cloned plasmid DNA into benign tumor cells has revealed that transforming constructs of the fos oncogene induce malignant conversion, whereas myc and adenovirus E1A oncogenes do not. The fos carcinomas do not express differentiation-specific epidermal markers and secrete proteases such as transin and urokinase, a set of characteristics previously noted for chemically induced skin carcinomas. Cultured normal epidermal cells, exposed to the v-ras and the v-fos oncogenes simultaneously, are malignantly transformed. Alone, the fos oncogene does not detectably alter the phenotype of normal keratinocytes. These studies indicate that a limited number of genes is involved in epidermal carcinogenesis.

Animals↗
PubMed1989

Comparison of octahydromezerein and mezerein as protein kinase C activators and as mouse skin tumor promoters.

Although mezerein resembles 12-O-tetradecanoylphorbol-13-acetate (TPA) in being a potent ligand for protein kinase C in vitro, its properties as a tumor promoter on mouse skin differ from those of TPA. Mezerein is a good second-stage promoter of papillomas, an inefficient complete promoter of papillomas, and an effective promoter of carcinomas. The mechanism and structural features responsible for the anomalous tumor promoting activity of mezerein are unknown. We have examined here the in vitro and in vivo activities of octahydromezerein (OHM) and compared them to those of TPA and mezerein. OHM was of interest because if it acted like mezerein it would afford a convenient route for radioactive labeling. Alternatively, if it functioned as a complete tumor promoter, it would implicate unsaturation as the critical structural feature of mezerein responsible for its altered promoting activity. Consistent with this latter possibility, we find that OHM was an effective complete tumor promoter for SENCAR mice. Moreover, the pattern and magnitude of papilloma induction, yielding a peak at 16-20 weeks followed by a decline by 30-32 weeks, resembled that for TPA; mezerein, in contrast, induced a gradual but steady increase in the number of papillomas which did not reach the OHM level by 32 weeks. The dosage of OHM for inducing a comparable degree of acute and chronic hyperplasia to that induced by mezerein was 3- to 10-fold higher. This difference agrees with the relative binding affinities to protein kinase C; the Ki for OHM was 2.7 nM, compared to 0.58 nM for mezerein.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Intracellular calcium alterations in response to increased external calcium in normal and neoplastic keratinocytes.

Normal keratinocytes proliferate when cultured in medium with 0.02-0.10 mM calcium and terminally differentiate when medium calcium is increased to greater than 0.1 mM. In contrast, neoplastic keratinocyte cell lines maintain the potential for continued cell renewal and survive when external calcium is increased. In order to determine whether elevation of extracellular calcium produced changes in intracellular free calcium (Cai) levels, Cai was measured in individual living keratinocytes by use of the fluorescent calcium probe fura-2. Most normal keratinocytes responded to increased extracellular calcium by a gradual 2- to 3-fold increase in Cai lasting for at least 28 min. A subpopulation displayed a sharp peak of Cai at 2 min. In contrast, the Cai level in neoplastic cells in either low or high calcium medium was 2- to 3-fold higher than that in normal cells, and all cells in the population showed a transient 4- to 9-fold elevation of Cai 2 min after external calcium was increased. Thus normal and neoplastic keratinocytes differ in the level of Cai under low calcium conditions and in their response to elevated external calcium. The regulation of Cai in keratinocytes may be important in determining their potential for terminal differentiation.

Animals↗