Anthony Anisowicz

IDENTIFICATION, CLONING, AND CHARACTERIZATION OF CYSTATIN M, A NOVEL CYSTEINE PROTEINASE INHIBITOR, DOWN-REGULATED IN BREAST CANCER

A novel human cystatin gene was identified in a differential display comparison aimed at the isolation of transcriptionally regulated genes involved in invasion and metastasis of breast cancer. Messenger RNAs from primary and metastatic tumor cells isolated from the same patient were compared. A partial cDNA was isolated that was expressed in the primary tumor cell line but not in the metastatic line. The full-length cDNA was cloned and sequenced, and the inferred amino acid sequence was found to encode a novel protein, which we named cystatin M, with 40% homology to human family 2 cystatins and similar overall structure. Cystatin M is expressed by normal mammary cells and a variety of human tissues. The mature cystatin M protein was produced in Escherichia coli as a glutathione S-transferase fusion protein using the pGEX-2T expression system and purified by affinity chromatography. The cystatin M fusion protein displayed inhibitory activity against papain. Native cystatin M protein of approximately 14.5 kDa is secreted and was immunoprecipitated from supernatants of mammary cell cultures using affinity-purified antisera raised against recombinant cystatin M. An N-glycosylated form of cystatin M of 20-22 kDa was co-immunoprecipitated and accounted for about 30-40% of total cystatin M protein. Both forms of native cystatin M also occurred intracellularly. Consistent with the mRNA differential expression, no cystatin M protein was detected in metastatic mammary epithelial tumor cells. Loss of expression of cystatin M is likely associated with the progression of a primary tumor to a metastatic phenotype.

Identification by differential display of alpha 6 integrin as a candidate tumor suppressor gene.

A new method of differential expression cloning called differential display (DD) has been used to screen for novel tumor suppressor genes involved in breast cancer. The screen is based on positive selection at the mRNA level for genes expressed in normal mammary epithelial cells but decreased or lost in corresponding tumor cells. A candidate tumor suppressor gene recovered by DD is integrin alpha‐6 (α6), a component of the heterodimeric integrin receptors α6β1 and α6β4. Loss of α6 expression was confirmed in total RNAs by Northern blot analysis and by immunostaining with α6 antibodies. Consistent with these cell culture findings, previous immunostaining of mammary tissue sections has identified decreased α6 protein expression during breast tumor progression. Southern blot analysis demonstrated that α6 gene is present in tumor cell lines, suggesting that re‐expression may be inducible by pharmacological intervention. The likelihood that α6 may have tumor suppressing activity is supported by growing evidence of a central role for integrins in transducing growth control and differentiation signals from growth factors and the extracellular matrix (ECM).—Sager, R., Anisowicz, A., Neveu, M., Liang, P., Sotiropoulou, G. Identification by differential display of alpha‐6 integrin as a candidate tumor suppressor gene. FASEB J. 7: 964‐970; 1993.

Genomic rearrangements in a mouse cell line containing integrated SV40 DNA

In the SV40-transformed mouse embryo fibroblast cell line SVT2/S, genomic rearrangements involving the SV40 DNA and flanking host sequences were identified by Southern blot hybridization using viral DNA as probe. No rearrangements of SV40 DNA integrated into nonpermissive mouse cells have been previously described. The standard arrangement found in the majority of subclones was mapped with 20 restriction enzymes, 10 of which cleave sites within the SV40 DNA. A single copy of a defective integrated viral genome is present, in which the late region is missing from about nucleotide 200 clockwise to about nucleotide 1750. The rest of the viral genome including the origin of replication and T antigen binding region is present and colinear with SV40 DNA, except for an internal repeat of about 1750 bp located between nucleotides 2750 and 4500. Rearrangements were found in 4 out of 20 random subclones of the parental SVT2/S cell line and 3 of the 4 continued to rearrange. The thioguanine-resistant cell line 281-1-4, derived from SVT2/S, remained stable on subculture but a chloramphenicol-resistant mutant, 107-6-4, derived from 281-1-4, was highly unstable. In 107-6-4, unique rearrangements were found in 6 of 31 subclones of a population that had undergone abut 25 doublings from a single-cell isolate. The high rate of rearrangement and the sporadic expression of rearrangement potential are characteristic of the transposable controlling elements discovered by McClintock.

GRO: A Novel Chemotactic Cytokine

Cytokines are small secreted regulatory peptides that act primarily in a paracrine fashion to provide a signalling system between different cell types. Although cytokine functions were initially associated with myeloid and lymphocytic responses to infection, current studies are demonstrating regulatory functions in growth and differentiation as well. Our studies with the GRO gene represent one of the first to describe a novel cytokine expressed by fibroblasts and epithelial cells as well as cells of the immune system, with regulatory functions both in growth and in the inflammatory response (1, 2, 3).

Re-expression of SPR1 in breast cancer cells by phorbol 12-myristate 13-acetate (PMA) or UV irradiation is mediated by the AP-1 binding site in the SPR1 promoter.

BackgroundInvasive tumor cells are characterized by multiple phenotypic changes as a result of the large number of cDNAs being differentially expressed in tumor cells compared to normal progenitors. Expression genetics focuses on changes at the RNA level with the aim of identifying functionally important genes whose aberrant expression in cancer cells is regulated at the level of transcription. These genes were named class II genes and are distinguished from class I genes, which are characterized by genomic mutations, deletions, or other alterations. Reversal of the tumor cell phenotype accompanying normalization of the expression of such genes may be exploited therapeutically if gene expression can be specifically modulated by drugs or other treatments. Considering that genes are coordinately regulated in complex networks, it is likely that the expression of multiple genes can be simultaneously modulated in tumor cells by drugs acting on the signal transduction pathway that regulates their expression. The SPR1 gene is associated with differentiation and its expression is down-regulated or inactivated in malignant cells. Analysis of the SPR1 promoter showed that down-regulation of SPR1 expression in breast tumor cells occurs at the level of transcription. SPR1 presents an example of class II genes, since its expression was up-regulated in tumor cells by phorbol 12-myristate 13-acetate (PMA) or by ultraviolet (UV) irradiation.Materials and MethodsThe SPR1 gene was identified by differential display on the basis of its reduced or absent expression in human breast tumor cell lines compared to normal mammary epithelial cell strains. Differential expression was confirmed by Northern blot analysis employing multiple normal and tumor cell lines. The promoter region −619 to +15 of the SPR1 gene was sequenced and analyzed by CAT assays, deletion analysis, and mutagenesis. Up-regulation of SPR1 expression by PMA and UV irradiation was monitored by Northern analysis and analyzed by CAT assays.ResultsThe mechanism of down-regulation of SPR1 expression in breast tumor cells was investigated. It was found that the −619 to +15 upstream promoter region is sufficient for SPR1 expression in normal breast cells, but it is transcriptionally silent in most breast tumor cell lines. By deletion analysis and mutagenesis, two upstream as-acting promoter elements were identified. Our data indicate that the AP-1 element located between −139 and −133 acts as a major enhancer of SPR1 transcription only in normal mammary epithelial cells but not in corresponding tumor cells, whereas the sequences flanking the AP-1 site do not affect its promoter enhancing activity. In addition, a transcriptional repressor was identified that binds unknown factor(s) and is active in both normal and tumor breast cells. Inhibitor function was mapped to a 3 5-bp element located from −178 to −139 upstream of the human SPR1 mRNA start site. The expression of SPR1 could be induced in the 21MT-2 metastatic breast tumor cell line by PMA treatment or by short UV irradiation via a transcriptional mechanism. AP-1 is the cis element mediating the transcriptional activation of SPR1 by PMA, which induces the expression of AP-1 factors in 21MT-2 cells. Mutation of the AP-1 site abolishes the induction of SPR1 expression by PMA.ConclusionsOur results demonstrate that loss of SPR1 expression in breast tumor cells results from impaired transactivation through the AP-1 site in the SPR1 promoter, as well as from the presence of a negative regulatory element active in both normal and tumor cells. Furthermore, our results provide a basis for therapeutic manipulation of down-regulated genes, such as SPR1, in human cancers.

Genetic analysis of tumorigenesis: XXXII. Localization of constitutionally amplifiedKRAS sequences to Chinese hamster chromosomes X and Y by in situ hybridization

The KRASgene is constitutionally amplified in the Chinese hamster. We have mapped the amplified sequences by in situ hybridization to two major sites on the X and Y chromosomes, Xq4 and Yp2. No autosomal site was detected despite a search under relaxed hybridization conditions. KRASDNA is amplified about 50-fold compared to a human cell line known to have a diploid number of KRASsequences, whereas mRNA expression is 5- to 10-fold lower than in normal human cells. While mRNA expression levels do not necessarily parallel gene copy number, the low expression level strongly suggests that the amplified sequences are transcriptionally silent. It is suggested that the amplified sequences arose from the original KRASgene on chromosome 8 and that the KRASsequences on the Y chromosome arose by X-Y recombination.

Down Regulation of Candidate Tumor Suppressor Genes in Breast Cancer

Changes in gene expression alter the tumorigenic potentialities of cancer cells either by increasing their oncogenec potential (oncogenes) or by decreasing it (tumor suppressor genes, here called TSGs) (1). The identification of oncogenes has been based on positive selection for loss of growth control, and has led to the recovery of numerous candidates, principally retroviral oncogenes, but the identification of TSGs based on negative selection has been meager. The need for positive selection schemes to identify TSGs has been a problem of outstanding importance in the molecular genetic analysis of cancer.