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Biomedical subjects

A Balmain

Publications and source records attributed to A Balmain.

At least 73 records · Page 4Linked to original sources
PubMed1992

Expression of simple epithelial cytokeratins in mouse epidermal keratinocytes harboring Harvey ras gene alterations.

Activation of a Harvey ras (H-ras) protooncogene is a frequent event associated with mouse epidermal carcinogenesis. We report that the transfection of a human H-ras oncogene into an immortalized mouse epidermal cell line (MCA3D) induces the anomalous expression of cytokeratins (CKs) 8 and 18 characteristic of simple epithelia. The comparison of various transfectant cell clones indicated a direct correlation between the levels of CK8 expression and the mutated H-ras p21s. The expression of simple epithelial CKs is also described in cell lines derived from mouse skin carcinomas (HaCa4, CarC) and in keratinocytes transformed in vitro by a chemical carcinogen (PDV, PDVC57), all of which contain altered H-ras genes. The induction of CK8 and CK18 occurs at the mRNA level and, although both CK8 and CK18 mRNAs are expressed, CK18 protein does not accumulate whereas CK8 is incorporated into intermediate filaments. Immunofluorescence studies show that the pattern of CK8 protein expression is heterogeneous; some cells express very low amounts of CK8, whereas others synthesize relatively high levels of this protein. However, selection of strongly CK8-positive cells was found in one case where a more malignant population of cells (PDVC57) was derived by tumor transplantation of PDV. Our results suggest that activation of a H-ras gene can alter the normal differentiation program of epidermal cells and that the ability to synthesize CK8 and CK18 could be related to tumor progression.

Animals↗
PubMed1992

Potential therapeutic targets in multistep oncogenesis.

There are three important considerations in the development of novel drug therapies: firstly, whether a suitable target can be accurately defined; secondly, does the target allow discrimination between normal cells and tumour cells; and thirdly, how easily could the tumour cells acquire resistance? This article will concentrate on two of the genetic alterations most commonly associated with multistep tumourigenesis, and discuss their potential as therapeutic targets with respect to the considerations above.

Animals↗
PubMed1992

Discordant transforming growth factor beta 1 RNA and protein localization during chemical carcinogenesis of the skin.

Transforming growth factor beta (TGF-beta) inhibits proliferation of normal keratinocytes, and this response is retained, to variable extents, in benign tumors of the skin (S. Haddow, D. J. Fowlis, K. Parkinson, R. J. Akhurst, and A. Balmain, Oncogene, 6: 1465-1470, 1991). To investigate the profile of TGF-beta biosynthesis during various stages of chemical carcinogenesis of the skin, we used a combination of ribonuclease protection assay, in situ hybridization with gene-specific probes for TGF-beta 1, -beta 2, and -beta 3, and immunohistochemistry with isoform-specific antibodies against TGF-beta 1. Following 12-O-tetradecanoylphorbol-13-acetate treatment of adult mouse skin, there was a rapid induction of TGF-beta 1 protein. Intracellular TGF-beta 1 protein was localized to suprabasal keratinocytes, and the extracellular form was localized predominantly to the dermis. Despite ubiquitous induction of TGF-beta 1 protein by 12-O-tetradecanoylphorbol-13-acetate in various mouse strains, we noted strain-specific differences in the quantitative induction of TGF-beta 1 RNA. Papillomas and carcinomas induced in vivo had elevated levels of TGF-beta 1 RNA within the basal keratinocyte compartment but did not contain significant levels of TGF-beta 1 protein within the tumor. We postulate that the tumor evades TGF-beta 1-controlled negative growth regulation by altered translational and/or posttranslational processing mechanisms of this growth factor. Levels of TGF-beta 2 and -beta 3 RNA were not elevated at any stage of chemical carcinogenesis of the skin.

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

Progression of squamous carcinoma cells to spindle carcinomas of mouse skin is associated with an imbalance of H-ras alleles on chromosome 7.

Analysis of benign and malignant mouse skin tumors had previously shown that amplification of a mutant H-ras allele or loss of the normal allele was generally seen only in high grade or spindle cell tumors. The normal:mutant ras gene dosage has been studied directly by polymerase chain reaction amplification of DNA derived from paraffin sections of carcinomas of defined histological types. Some tumors had virtually no signal corresponding to the normal allele and these were invariably spindle cell carcinomas. In four cases where both squamous and spindle cell components could be identified within the same tumor the spindle cell component had a higher mutant:normal gene ratio. Additional experiments on cell lines derived from squamous or spindle cell tumors have demonstrated a good correlation between the ratio of normal:mutant ras and the degree of invasiveness of the cells in in vitro assays.

Alleles↗
PubMed1991

Structural analysis of the mouse c-Ha-ras gene promoter.

Previous studies have demonstrated that the mouse c-Harvey ras proto-oncogene (c-Ha-ras) promoter sequences are GC rich and contain several potential transcription factor SP1 binding sites. We investigated the endonuclease hypersensitivity of this region in nuclei in vitro and whole mouse tissues in vivo and identified a very strong, ubiquitous hypersensitive site covering the proximal promoter sequences. Footprint protection studies using nuclear extracts from various cell types including fibroblasts, erythroid cells, and both normal and transformed epithelial cells revealed a consistent protein-binding pattern. Five protein binding sites were observed, four of which correlated with potential SP1 binding sites. Competition experiments using an oligonucleotide corresponding to a consensus SP1 binding site confirmed that these sequences were indeed bound by the SP1 (or SP1-like) trans-acting factor. In addition, no differences were observed between the footprint patterns obtained using extracts from cells of different lineages or between normal and transformed epithelial cells carrying activated ras genes. The controlling elements responsible for differential c-Ha-ras transcription between cell types or at different stages of carcinogenesis therefore probably lie in other regions of the gene.

Animals↗
PubMed1991

Comparison of ras activation during epidermal carcinogenesis in vitro and in vivo.

Mouse epidermal cells have frequently been used to study the role of ras oncogenes in transformation in vivo and in vitro. After initiation with dimethylbenzanthracene (DMBA) in vivo, greater than 90% of the papillomas arising show the same A:T----T:A transversion at codon 61 of the H-ras gene, presumed to be the initiating event. On the other hand, initiation of epidermal cells in culture with carcinogens, followed by selection of initiated cells by resistance to calcium-induced differentiation, does not in general lead to the isolation of clones carrying mutant ras genes. Some other aspects of tumour progression in vivo can be reproduced using epidermal cells in culture: a rare DMBA transformant carrying the codon 61 mutation and expressing a 2:1 ratio of normal to mutant ras alleles gave rise upon transplantation to a more aggressive line in which the ratio of normal to mutant H-ras genes (and p21 products) was reversed. Similar alterations in ras gene dosage have been seen during progression of papillomas to carcinomas in vivo. We conclude that the mechanisms of initiation in vitro may differ substantially from in vivo, and depend on the particular culture conditions used. Moreover, the effects of mutant H-ras expression in mouse epidermal cells are variable depending on the genetic background of the cell.

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

Genetic changes during mouse skin tumorigenesis.

This paper describes specific genetic changes involving chromosome 7 in mouse skin tumors, the most important consequence of which appears to be an alteration in the allelic balance of normal and mutant H-ras genes. The use of restriction-fragment-length polymorphisms in F1 hybrid mice demonstrates that trisomy of chromosome 7 is an early event preceding papilloma formation, and further events, such as mitotic recombination, seem to occur during progression to malignant carcinomas. There is some evidence of a tumor-suppressor locus situated on chromosome 7.

Alleles↗
PubMed1991

Loss of heterozygosity and mutational alterations of the p53 gene in skin tumours of interspecific hybrid mice.

Functional alterations or loss of tumor-suppressor genes are an important feature of neoplastic progression in humans. The employment of suitable animal model systems would greatly facilitate the detection and manipulation of such genes. We describe here an experimental approach to this problem based on the analysis of skin tumors induced in F1 hybrids between Mus musculus and Mus spretus mice. The results show that loss of heterozygosity on chromosome 11 occurred in 4/13 mouse skin carcinomas, but not in premalignant papillomas. Since the murine p53 gene is located on this chromosome, immunoprecipitation and DNA-sequencing studies were carried out on tumorigenic cell lines and primary tumor DNA respectively to determine the status of p53 alleles. These studies revealed the presence of p53 mutations, both frameshifts and missense, some of which are identical to those found in human tumors. Loss of normal p53 function is found in well-differentiated squamous-cell carcinomas and thus does not appear to be directly responsible for further progression to an undifferentiated spindle cell phenotype.

Amino Acid Sequence↗
PubMed1991

Functional loss of tumour suppressor genes in multistage chemical carcinogenesis.

Studies of multistage carcinogenesis in mouse skin have provided many of the early concepts of tumour initiation, promotion and progression. Genetic approaches have led to the identification of a number of mutational alterations in proto-oncogenes and tumour suppressor genes which take place at specific stages of carcinogenesis in this particular system. Initiation involves, at least in a proportion of tumours, mutational activation of the cellular H-ras proto-oncogene. Trisomy of chromosome 7, which develops during the premalignant clonal expansion phase, possibly as a consequence of tumour promoter treatment, is followed by further alterations on chromosome 7 which lead to a relative increase in the expression of mutant ras alleles. The p53 tumour suppressor gene undergoes mutational alteration and loss of heterozygosity in a proportion of squamous carcinomas but this particular gene does not appear to be involved in the further transition of squamous carcinomas to highly undifferentiated spindle cell tumours. The latter transition appears to be a recessive event which can be complemented by fusion with cells at earlier stages of malignancy. Mouse skin carcinogenesis therefore continues to provide invaluable information on the nature of the genetic and biological transitions which occur during the step-wise progression of normal cells to malignancy.

Animals↗
PubMed1991

Loss of growth control by TGF-beta occurs at a late stage of mouse skin carcinogenesis and is independent of ras gene activation.

The relationship between the expression of a mutant ras gene in epithelial cells and loss of responsiveness to the negative effects of transforming growth factor beta (TGF-beta) is presently unclear. We have investigated this question using a series of cell lines derived from benign and malignant mouse skin tumours which express mutant forms of the H-ras gene. Immortalised, non-tumorigenic mouse epidermal cells respond to TGF-beta by cessation of growth, whereas in a series of malignant carcinoma lines the response was substantially reduced. Introduction of a mutant H-ras gene into the immortalised cells did not lead to any appreciable change in TGF-beta responsiveness, suggesting that initiation of carcinogenesis by ras mutation does not directly alter growth control by this pathway. Of two non-tumorigenic papilloma lines tested which had mutant H-ras genes, one retained complete sensitivity to TGF-beta, whereas the other showed a similar response to carcinomas. We conclude that growth control by TGF-beta is lost at a relatively late stage of carcinogenesis in this system, and is independent of ras gene activation.

Animals↗
PubMed1991

Transformation of mouse skin endothelial cells in vivo by direct application of plasmid DNA encoding the human T24 H-ras oncogene.

Plasmid DNA containing the human T24 H-ras oncogene, with or without viral transcriptional enhancer sequences, was applied to scarified mouse skin, followed by multiple treatments with the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate. This resulted in the formation of vasoformative tumours histologically characterized as lymphangiosarcomas. All of the animals treated developed cystic fluid-filled swellings. Polymerase chain reaction analysis revealed the presence of human H-ras sequences within the cystic fluid from 3 out of 4 swellings. An endothelial cell line established from the cystic fluid removed from one of these swellings was found to contain human H-ras sequences and to express the mutant human p21ras. Injection of the cell line into nude mice, or adult syngeneic mice, resulted in the formation of aggressive angiosarcomas. Further experiments showed that 12-O-tetradecanol-phorbol-13-acetate promotion is not required for tumour formation and would appear to reduce the yield of tumours. These results indicate that a single application of the human H-ras oncogene is sufficient to induce endothelial cell transformation in vivo, even in the absence of any further promotional stimulus.

Animals↗
PubMed1990

Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter.

The promoter region of the suprabasal keratin 10 gene has been used to direct expression of a mutant human Harvey-ras oncogene to the differentiating cells of the mouse epidermis. Transgenic animals develop hyperkeratosis of the skin and forestomach--the two sites known to express high levels of the keratin 10 polypeptide in vivo. Papillomas subsequently develop on the skin surface, initially at sites subject to biting or scratching such as the base of the tail or behind the ears. The results suggest that the "second event" involved in tumor development in these transgenic animals is the local induction of a mild wounding stimulus. Furthermore, because the H-ras transgene is expressed in suprabasal cells, it appears that cells which have left the stem cell compartment can be induced to form at least benign tumors in vivo.

Animals↗
PubMed1990

Genetic changes in skin tumor progression: correlation between presence of a mutant ras gene and loss of heterozygosity on mouse chromosome 7.

Initiation of tumorigenesis in mouse skin can be accomplished by mutagenesis of the H-ras gene by treatment with chemical carcinogens. A mouse model system has been developed to study the additional genetic events that take place during tumor progression. Skin carcinomas were induced in F1 hybrid mice exhibiting restriction fragment length polymorphisms at multiple chromosomal loci. Analysis of loss of heterozygosity in such tumors showed that imbalance of alleles on mouse chromosome 7, on which the H-ras gene is located, occurs very frequently in skin carcinomas. The chromosomal alterations detected, which included both nondisjunction and mitotic recombination events, were only seen in tumors that have activated ras genes. We conclude that gross chromosomal alterations that elevate the copy number of mutant H-ras and/or lead to loss of normal H-ras are a consistent feature of mouse skin tumor development.

Alleles↗
PubMed1990

Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression.

A number of mouse skin tumors initiated by the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz[a]anthracene (DMBA) have been shown to contain activated Ha-ras genes. In each case, the point mutations responsible for activation have been characterized. Results presented demonstrate the carcinogen-specific nature of these ras mutations. For each initiating agent, a distinct spectrum of mutations is observed. Most importantly, the distribution of ras gene mutations is found to differ between benign papillomas and carcinomas, suggesting that molecular events occurring at the time of initiation influence the probability with which papillomas progress to malignancy. This study provides molecular evidence in support of the existence of subsets of papillomas with differing progression frequencies. Thus, the alkylating agents MNNG and MNU induced exclusively G ---- A transitions at codon 12, with this mutation being found predominantly in papillomas. MCA initiation produced both codon 13 G ---- T and codon 61 A ---- T transversions in papillomas; only the G ---- T mutation, however, was found in carcinomas. These findings provide strong evidence that the mutational activation of Ha-ras occurs as a result of the initiation process and that the nature of the initiating event can affect the probability of progression to malignancy.

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

Analysis of the HMGI nuclear proteins in mouse neoplastic cells induced by different procedures.

Four malignant tumors induced in mouse by different experimental procedures were compared as regards their high-mobility-group (HMG) proteins. All tumors showed the complete set of three HMG proteins which we call HMGI-C, I-D, and I-E. The presence of the three HMGI proteins is a characteristic of the transformed phenotype regardless of whether the tumor was chemically, virally, or spontaneously derived. However, the level of expression of the HMGI proteins is not constant in the four tumors. Using reverse-phase HPLC, the individual HMGI proteins were isolated from the spontaneously derived tumor (Lewis lung carcinoma) and shown by amino acid analysis to be similar to those previously obtained from a tumor grown in nude mice by inoculation of in vitro-transformed cells.

Amino Acids↗