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M L Breitman

Publications and source records attributed to M L Breitman.

At least 55 records · Page 3Linked to original sources
PubMed1989

Multiple regulatory elements of the murine gamma 2-crystallin promoter.

Crystallins are the major water-soluble proteins of the vertebrate eye lens. These lens-specific proteins are encoded by several multi-gene families whose expression is differentially regulated during development. Our previous studies showed that the mouse gamma 2-crystallin promoter is active on transfection into lens-explant cultures derived from 14-day-old chick embryos but not on transfection into a variety of non-lens cells. In this study, transient expression data show that a sequence of 226 nucleotides upstream from the transcription start site is sufficient for activity of this promoter in the chicken lens cells. This sequence can be further divided into two domains, A and B, both of which are required for promoter function. Domain A (nucleotide -68 to -18) contains the TATA box and sequence motifs that are conserved in all gamma-crystallin promoters. Domain B (-226 to -120) consists of three regions. One of these regions contains an element with dyad symmetry and a sequence similar to the octamer motif. The second region contains an enhancer core consensus sequence. Two "enhancer-like" activities have been detected, one in Domain B and a second in a more distal region (-392 to -278) that does not appear to be required for promoter activity in transfection assays.

Animals↗
PubMed1989

A rabbit lens epithelial cell line supports expression of an exogenous crystallin gene characteristic of lens fiber cell differentiation.

Cell lines derived from the lens generally fail to maintain synthesis of crystallins in long-term culture. Here we demonstrate that the N/N1003A line of undifferentiated lens epithelial cells, derived from a newborn rabbit, does not produce detectable levels of alpha-, beta- or gamma-crystallin transcripts, yet is capable of supporting the transient expression of the mouse gamma 2-crystallin promoter, a promoter which is active only in terminally differentiated lens fiber cells in vivo. Analysis of a set of deletion constructs suggested that sequences required for activity of the mouse promoter in N/N1003A cells are similar, but not identical, to those previously shown to be essential in primary chick embryo lens explants. Therefore, these results suggest that different transcriptional factors may be capable of supporting lens-specific activity of the mouse gamma 2 promoter. In addition, this cell line, N/N1003A, should be useful for investigations on the elements regulating gamma-crystallin gene expression.

Animals↗
PubMed1989

Analysis of lens cell fate and eye morphogenesis in transgenic mice ablated for cells of the lens lineage.

Transgenic mice carrying the diphtheria toxin A gene driven by mouse gamma 2-crystallin promoter sequences manifest microphthalmia due to ablation of fiber cells in the ocular lens. Here we map ablation events in the lens by crossing animals hemizygous for the ablation construct with transgenic mice homozygous for the in situ lacZ reporter gene driven by identical gamma 2-crystallin promoter sequences. By comparing the spatial distribution of lacZ-expressing cells and the profile of gamma-crystallin gene expression in the lenses of normal and microphthalmic offspring, the contributions of specific cell types to lens development were examined. The results suggest that phenotypically and developmentally distinct populations of lens fiber cells are able to contribute to the lens nucleus during organogenesis. We also show that dosage of the transgene and its site of integration influence the extent of ablation. In those mice homozygous for the transgene and completely lacking cells of the lens lineage, we show that the sclera, cornea, and ciliary epithelium are reduced in size but, otherwise, reasonably well formed. In contrast, the anterior chamber, iris, and vitreous body are not discernible while the sensory retina is highly convoluted and extensively fills the vitreous chamber.

Animals↗
PubMed1989

Oncogenes conferring metastatic potential induce increased branching of Asn-linked oligosaccharides in rat2 fibroblasts.

Increased -GlcNA beta-6 Man alpha 1-6 Man beta 1- branching of Asn-linked oligosaccharides has been observed in a number of rodent and human tumor cell lines and the structures have been correlated with enhanced metastatic potential of murine tumor cells. Here we have compared the malignant potential and levels of beta 1-6-branched oligosaccharides in rat 2 fibroblast transfected with the cytoplasmic tyrosine kinase v-fps/fes, the activated GTPase T24 H-ras, and the nuclear oncogene c-myc. Rat2 cells transfected with activated c-myc were non-tumorigenic in nude mice and did not show elevated levels of beta 1-6 branched oligosaccarides, whereas transfectants carrying H-ras or v-fps were tumorigenic and generally exhibited metastatic potential which was associated with increased beta 1-6 branching. Enhanced expression of beta 1-6 branched oligosaccharides did not correlate with increased ratios of UDP-HexNAc to UDP-Hex, but was accompanied by elevated GlcNAc-transferase V activity, increased sensitivity of the cells to the toxic effects of leukoagglutinin, and an altered intracellular distribution of beta 1-6-branched oligosaccharides as visualized by fluorescent lectin staining. These results provide information on the quantitative and qualitative relationships between oncogene expression and cellular glycosylation and suggest that the ability of an oncogene to confer metastatic potential may be related to its ability to induce increased branching of Asn-linked oligosaccharides.

Animals↗
PubMed1989

Asparagine-linked oligosaccharides associated with metastatic cancer.

Malignant transformation and tumor progression are often accompanied by structural changes in the carbohydrate components of glycoproteins and glycolipids. Studies with tumor cell glycosylation mutants and specific inhibitors of glycosylation indicate that expression of sialylated and beta (1-6) branched asparagine (Asn)-linked oligosaccharides are required for tumor cell invasion and metastasis. The initiation of the beta (1-6) antenna appears to be oncodevelopmentally regulated and may be associated with the aberrant expression of blood group and embryonic carbohydrate sequences in Asn-linked oligosaccharides of tumors. Although the function of these oligosaccharides in the metastatic process remains unclear, there is some evidence that expression of these structures on certain cell-surface glycoproteins may reduce tumor cell adhesion to extracellular matrix. These observations suggest that low-toxicity drugs that inhibit the biosynthesis of these structures may be useful in the treatment of metastatic cancers.

Animals↗
PubMed1988

Genetic evidence for progressive selection and overgrowth of primary tumors by metastatic cell subpopulations.

We have exploited random insertions of transfected DNA as unique clonotypic markers to follow cell lineages during primary and metastatic tumor growth of a mouse mammary adenocarcinoma, SP1. Southern analysis was undertaken of primary solid tumors and metastases obtained after injection of a pooled population of individual SP1 transfectants, or reconstituted mixtures of genetically marked metastatic and unmarked nonmetastatic cells. Here we provide evidence for the reproducible selection and eventual overgrowth of primary tumors by genotypically distinct metastatic clones, thereby illustrating that late-state, advanced primary tumors can evolve to become biologically similar, or even identical, to distant metastases. The selective growth advantage of metastatic cells within primary tumors was shown to occur despite the fact that tumors generated by both metastatic and nonmetastatic SP1 cell populations grew at comparable growth rates when injected and analyzed separately. The extent of the local growth advantage manifested by individual metastatic clones varied considerably, from 5- to 50-fold. Clonal overgrowth was also observed whether the tumor cells were injected ectopically, or orthotopically (i.e., into the mammary fat). This type of experimental approach should provide new insights into the dynamics of tumor progression and metastasis, the lineage relationship of primary tumors to metastases, the influence of clonal interactions on tumor behavior, and the physiological changes which are causative of malignant disease.

Adenocarcinoma↗
PubMed1988

Clonal changes in tumours during growth and progression evaluated by southern gel analysis of random integrations of foreign DNA.

We have exploited random integrations of foreign DNA as a means of genetically tagging tumour cell populations with which to analyse the clonal evolution of tumour growth in vivo. Transfection of a non-metastatic mouse mammary carcinoma called SP1 (or a metastatic variant, SP1HU9L) with the pSV2neo plasmid or retrovirus vector infection with a "clipped-wing' vector (delta p delta eMoTN) was used to generate large numbers of uniquely marked tumour cell clones in single-step selections. The basic approach was to pool large numbers of independently marked transfectants or infectants, inject these cells into mice and analyse the resulting primary tumours and/or metastases later. Overgrowth or derivation of tumour masses by a limited number of clones could be detected by Southern gel analysis. The main findings were: (i) injection of pooled populations containing large numbers of uniquely marked cell clones (up to several thousand) invariably resulted in advanced primary tumours that contained a very limited number of clones, and in some cases only one easily detectable clone; (ii) primary tumours could be overgrown within six weeks by the progeny of the same single metastatic clone when the inoculum contained 1-10% metastatic cells, which suggests that metastatic SP1 cells have a selective growth advantage in primary tumours as well as for metastatic spread; and (iii) spontaneous lung metastases were clonal or biclonal at the time of analysis. The results show that spontaneous metastases can develop from a genetically distinct subpopulation of cells in a non-random (i.e. selective) manner. Because primary tumours can become overgrown by the progeny of a metastatic clone, results of any comparison of the properties of a primary tumour with a distant metastasis could be affected by the stage at which the primary tumour is removed and analysed.

Animals↗
PubMed1988

Clonal dominance of primary tumours by metastatic cells: genetic analysis and biological implications.

A new method is described for analysing the clonal evolutionary dynamics of tumour growth and the lineage relationship of primary tumours to their metastases. It exploits random integrations of transfected plasmid or retroviral infected (proviral) DNA as a means of generating very large numbers of uniquely marked cell clones in a single-step selection whose fates can then be tracked during progressive tumour growth. Using a mouse breast adenocarcinoma we undertook experiments in which syngeneic mice were injected with a mixture of very large numbers of uniquely marked cell clones, only one or a few of which were metastatic, or with reconstituted mixtures containing a genetically tagged metastatic clone with an excess of non-marked non-metastatic tumour cells. Among the results we summarize is the finding that spontaneous metastases developed in a non-random fashion from genotypically distinct cell clones. They were clonal or biclonal at the time of analysis. We also found that the progeny of a single metastatic clone could eventually overgrow the primary tumour. Thus malignant (metastatic) cells may manifest a striking growth advantage within the primary tumour site as well as for dissemination and growth at distant, secondary sites. As a result, late-stage advanced primary tumours, if left intact, may evolve to become biologically similar or equivalent to distant metastases. This 'clonal dominance' phenomenon can reconcile many of the discrepant experimental findings with respect to the putative selective nature of metastatic phenotype. Furthermore, it has important consequences for understanding one source of biological variability in experiments in which different primary tumours are compared to each other or to metastases; it also has implications for theories regarding the clonal origin of neoplasms, and for the physiological and biochemical changes that cause malignant disease.

Animals↗
PubMed1987

Genetic ablation: targeted expression of a toxin gene causes microphthalmia in transgenic mice.

Lineage-specific regulatory elements can be used to direct expression of a variety of genes to specific tissues in transgenic mice. If the hybrid constructs contain a gene encoding a cytotoxic gene product, then genetic ablation of a specific cell lineage can be achieved. We have generated six transgenic mice by introducing into fertilized eggs the mouse gamma 2-crystallin promoter fused to the coding region of the diphtheria toxin A-chain gene. Three of these mice and all the transgenic offspring analyzed were microphthalmic. The lenses of these mice displayed considerable heterogeneity: some were almost normal morphologically but reduced in size, whereas others were grossly aberrant and deficient in nuclear fiber cells. These studies indicate that programmed ablation of specific cell types can be stably transmitted through the germ line.

Animals↗
PubMed1987

Beta 1-6 branching of Asn-linked oligosaccharides is directly associated with metastasis.

Neoplastic transformation has been associated with a variety of structural changes in cell surface carbohydrates, most notably increased sialylation and beta 1-6-linked branching of complex-type asparagine (Asn)-linked oligosaccharides (that is, -GlcNAc beta 1-6Man alpha 1-6Man beta 1-). However, little is known about the relevant glycoproteins or how these transformation-related changes in oligosaccharide biosynthesis may affect the malignant phenotype. Here it is reported that a cell surface glycoprotein, gp 130, is a major target of increased beta 1-6-linked branching and that the expression of these oligosaccharide structures is directly related to the metastatic potential of the cells. Glycosylation mutants of a metastatic tumor cell line were selected that are deficient in both beta 1-6 GlcNAc transferase V activity and metastatic potential in situ. Moreover, induction of increased beta 1-6 branching in clones of a nonmetastatic murine mammary carcinoma correlated strongly with acquisition of metastatic potential. The results indicate that increased beta 1-6-linked branching of complex-type oligosaccharides on gp 130 may be an important feature of tumor progression related to increased metastatic potential.

Animals↗
PubMed1987

In situ detection of beta-galactosidase in lenses of transgenic mice with a gamma-crystallin/lacZ gene.

Transgenic mice carrying the gamma 2-crystallin promoter fused to the coding region of the bacterial lacZ gene were generated. The offspring of three founder mice expressed high levels of the enzyme solely in the central nuclear fiber cells of the lens as measured by an in situ assay for the detection of beta-galactosidase activity. These results suggest that gamma 2-crystallin sequences between -759 to +45 contain essential information required for appropriate tissue-specific and temporal regulation of the mouse gamma 2-crystallin gene. In a broader context, this study also demonstrates the utility of beta-galactosidase hybrid gene constructs for monitoring the activity of gene regulatory elements in transgenic mice.

Animals↗
PubMed1987

Differential regulation of gamma-crystallin genes during mouse lens development.

Using gene-specific probes derived from four mouse gamma-crystallin cDNAs, we have examined the regulation of different members of the mouse gamma-crystallin gene family during lens development. Our analysis revealed that, while the different gamma-crystallin genes appear to be coordinately activated during embryogenesis, the steady-state levels of their corresponding transcripts are differentially regulated, resulting in variations in the relative abundance of individual species at different stages of development. This complex pattern of gene regulation presumably accounts for one of the mechanisms determining the spatial distribution of different gamma-crystallins within the lens.

Aging↗
PubMed1987

A locus for a human hereditary cataract is closely linked to the gamma-crystallin gene family.

Within the human gamma-crystallin gene cluster polymorphic Taq I sites are present. These give rise to three sets of allelic fragments from the gamma-crystallin genes. Together these restriction fragment length polymorphisms define eight possible haplotypes, three of which (Q, R, and S) were found in the Dutch and English population. A fourth haplotype (P) was detected within a family in which a hereditary Coppock-like cataract of the embryonic lens nucleus occurs in heterozygotes. Haplotype P was found only in family members who suffered from cataract, and all family members who suffered from cataract had haplotype P. The absolute correlation between the presence of haplotype P and cataract within this family shows that the gamma-crystallin gene cluster and the locus for the Coppock-like cataract are closely linked [logarithm of odds (lod) score of 7.58 at its maximum at phi = 0]. This linkage provides genetic evidence that the primary cause of a cataract in humans could possibly be a lesion in a crystallin gene.

Cataract↗
PubMed1987

Alteration of the tumorigenic and metastatic properties of neoplastic cells is associated with the process of calcium phosphate-mediated DNA transfection.

During the course of studies designed to assess the effect of human Ha-ras gene expression on the malignant behavior of transfected mouse tumor cells we noted that the process of Ca3(PO4)2-mediated DNA transfection was itself associated with profound alterations in tumorigenic or metastatic behavior. The cell line used as a recipient for these studies was a tumorigenic nonmetastatic CBA/J mouse mammary adenocarcinoma line called SP1. When cotransfected with plasmids containing the neo gene (pSV2neo) and the activated Ha-ras gene (pT24-c3), cells from the pooled (5-10 colonies) G418-resistant colonies gave rise to spontaneous lung metastases in 85% of mice after subcutaneous inoculation. However, we noted that 17% of control mice inoculated with G418-resistant pSV2neo-transfected SP1 cells also had lung metastases and that this number approached 100% as the inoculum comprised a greater pool size (50-100 colonies). When cell lines established from isolated pSV2neo-transfected colonies were examined, 3/16 were found to be metastatic. We also found that 3/16 clones grew slowly, or not at all, in CBA/J mice, whereas they grew readily in athymic (nude) mice. The increase in immunogenicity of two out of three of these latter clones was accompanied by expression of the class I H-2Dk major histocompatibility complex antigen that was not detectable in the parental SP1 cells. At least some of these results would appear to be due to exposure to Ca3(PO4)2 alone, as we found that it resulted in 5/20 (25%) clones manifesting metastatic properties. Our results suggest that heritable changes in malignant behavior of transfected tumor cells can be observed at high frequency subsequent to the process of Ca3(PO4)2-mediated DNA transfection, and these changes may be brought about in part by inherited disturbances in expression of recipient cell genes.

Adenocarcinoma↗
PubMed1987

Aging effects of vitamin C on a human lens protein produced in vitro.

Human lens gamma-crystallin obtained from the expression of a gene construct stably integrated into mouse L cells was incubated with ascorbate in the presence of iron and oxygen. The resulting oxidation of the gamma-crystallin led to more acidic species of this protein. These alterations were similar to the changes seen with aging in the human lens. The results suggest that oxidation of lens crystallins may be responsible for the changes seen on aging and cataract development and that ascorbate may contribute to these alterations.

Aging↗
PubMed1987

The mouse eye lens obsolescence (Elo) mutant: studies on crystallin gene expression and linkage analysis between the mutant locus and the gamma-crystallin genes.

Previous studies showed that the mouse Elo (eye lens obsolescence) mutation is located on chromosome 1, at a site near the Len-1 locus, which is defined by a set of polymorphic gamma-crystallin proteins. To investigate further the relationship between Elo and the gamma-crystallins, we have examined the steady-state levels of gamma-crystallin transcripts in normal and mutant eyes and analyzed the linkage relationship between the Elo locus and the gamma-crystallin genes. Our data showed that, while gamma-crystallin mRNA levels are preferentially reduced in the mutant eyes, the mutation does not seem to map within the gamma-crystallin gene cluster. The distance between Elo and the gamma 6 gene (the most proximal gamma-crystallin gene member) is estimated to be 1.4 +/- 0.9 cM, whereas that between gamma 6 and the distantly linked gamma 2 gene is 2.7 +/- 1.3 cM. Our data also suggest the possibility of recombination hot spots with the gamma-crystallin gene cluster.

Alleles↗