Genomic structure, chromosomal localization and expression profile of a novel melanoma differentiation associated (mda-7) gene with cancer specific growth suppressing and apoptosis inducing properties.

Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-beta plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-beta+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-beta+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.

AP-1 and C/EBP transcription factors contribute to mda-7 gene promoter activity during human melanoma differentiation.

Treatment of human melanoma cells with a combination of recombinant fibroblast interferon (IFN-beta) and the protein kinase C (PKC) activator mezerein (MEZ) causes a rapid and irreversible suppression in growth and terminal cell differentiation. Temporal subtraction hybridization combined with random clone selection, reverse Northern hybridization, high throughput microchip cDNA array screening, and serial cDNA library arrays permit the identification and cloning of genes that are differentially expressed during proliferative arrest and terminal differentiation in human melanoma cells. A specific melanoma differentiation associated (mda) gene, mda-7, exhibits reduced expression as a function of melanoma progression from melanocyte to metastatic melanoma. In contrast, treatment of metastatic melanoma cells with IFN-beta + MEZ results in expression of mda-7 mRNA and protein. To evaluate the mechanism underlying the differential expression of mda-7 as a function of melanoma progression and induction of growth arrest and differentiation in human melanoma cells the promoter region of this gene has been isolated from a human placental genomic library and characterized. Sequence analysis by GCG identifies multiple recognition sites for the AP-1 and C/EBP transcription factors. Employing a heterologous mda-7 luciferase gene reporter system, we demonstrate that ectopic expression of either AP-1/cJun or C/EBP can significantly enhance expression of the mda-7 promoter in melanoma cells. In contrast, a dominant negative mutant of cJun, TAM67, is devoid of promoter-enhancing ability. Western blot analyses reveals that cJun and the C/EBP family member C/EBP-beta are physiologically relevant transcription factors whose expression corresponds with mda-7 mRNA expression. Electrophoretic mobility shift assays (EMSA) performed using nuclear protein extracts from terminally differentiated human melanoma cells document binding to regions of the mda-7 promoter that correspond to consensus binding sites for AP-1 and C/EBP. These results provide further mechanistic insights into the regulation of the mda-7 gene during induction of terminal cell differentiation in human melanoma cells.

Regulation of mda-7 gene expression during human melanoma differentiation.

Induction of irreversible growth arrest and terminal differentiation in human melanoma cells following treatment with recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ) results in elevated expression of a specific melanoma differentiation associated gene, mda-7. Experiments were conducted to define the mechanism involved in the regulation of mda-7 expression in differentiating human melanoma cells. The mda-7 gene is actively transcribed in uninduced HO-1 human melanoma cells and the rate of transcription of mda-7 is not significantly enhanced by treatment with IFN-beta, MEZ or IFN-beta+MEZ. The high basal activity of the mda-7 promoter in uninduced melanoma cells and the absence of enhancing effect upon treatment with differentiation inducers is corroborated by transfection studies using the promoter region of mda-7 linked to a luciferase reporter gene containing the SV40 polyadenylation signal sequence. RT - PCR analysis detects the presence of low levels of mda-7 transcripts in uninduced and concomitant increases in differentiation inducer treated HO-1 cells. However, steady-state mda-7 mRNA is detected only in IFN-beta+MEZ and to a lesser degree in MEZ treated cells. We show that induction of terminal differentiation of HO-1 cells with IFN-beta+MEZ dramatically increases the half-life of mda-7 mRNA while treatment with cycloheximide results in detectable mda-7 mRNA in control and inducer treated cells. These observations confirm constitutive activity of the mda-7 promoter in HO-1 cells irrespective of differentiation status suggesting posttranscriptional processes as important determinants of mda-7 expression during terminal differentiation. The 3' UTR region of mda-7 contains AU-rich elements (ARE) that contribute to rapid mda-7 mRNA turnover during proliferation and reversible differentiation, a process controlled by a labile protein factor(s). Substitution of the SV40 polyadenylation signal sequence in the luciferase reporter plasmid with the mda-7-ARE-3'-UTR renders the Luciferase message unstable when expressed in proliferating and reversibly differentiated melanoma cells. In contrast, the luciferase message is stabilized when the mda-7-ARE-3'-UTR construct is expressed in terminally differentiated HO-1 cells. These results provide compelling evidence that mda-7 expression during terminal differentiation in human melanoma cells is regulated predominantly at a posttranscriptional level.

The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice.

A differentiation induction subtraction hybridization strategy is being used to identify and clone genes involved in growth control and terminal differentiation in human cancer cells. This scheme identified melanoma differentiation associated gene-7 (mda-7), whose expression is up-regulated as a consequence of terminal differentiation in human melanoma cells. Forced expression of mda-7 is growth inhibitory toward diverse human tumor cells. The present studies elucidate the mechanism by which mda-7 selectively suppresses the growth of human breast cancer cells and the consequence of ectopic expression of mda-7 on human breast tumor formation in vivo in nude mice. Infection of wild-type, mutant, and null p53 human breast cancer cells with a recombinant type 5 adenovirus expressing mda-7, Ad.mda-7 S, inhibited growth and induced programmed cell death (apoptosis). Induction of apoptosis correlated with an increase in BAX protein, an established inducer of programmed cell death, and an increase in the ratio of BAX to BCL-2, an established inhibitor of apoptosis. Infection of breast carcinoma cells with Ad.mda-7 S before injection into nude mice inhibited tumor development. In contrast, ectopic expression of mda-7 did not significantly alter cell cycle kinetics, growth rate, or survival in normal human mammary epithelial cells. These data suggest that mda-7 induces its selective anticancer properties in human breast carcinoma cells by promoting apoptosis that occurs independent of p53 status. On the basis of its selective anticancer inhibitory activity and its direct antitumor effects, mda-7 may represent a new class of cancer suppressor genes that could prove useful for the targeted therapy of human cancer.

Programmed DNA degradation and nucleolar biogenesis occur in distinct organelles during macronuclear development in Tetrahymena.

Programmed DNA rearrangements, including DNA degradation, characterize the development of the soma from the germline in a number of developmental systems. Pdd1p (programmed DNA degradation 1 protein), a development-specific polypeptide in Tetrahymena, is enriched in developing macronuclei (anlagen) and has been implicated in DNA elimination and nucleolar biogenesis. Here, immunocytochemistry and fluorescent in situ hybridization (FISH) were employed to follow Pdd1p and two nucleolar markers (Nopp52 and rDNA) during macronuclear development. Both Pdd1p and Nopp52 localize to subnuclear structures, each of which resemble nucleoli. However, while true nucleoli form and persist during development, Pdd1p-positive structures are only present for a brief period of macronuclear differentiation. Accordingly, two distinct organelles can be recognized in anlagen: (1) Pdd1p-positive structures, which lack Nopp52 and rDNA, and (2) developing nucleoli which contain rDNA and Nopp52 but lack Pdd1p. Taken together with recent data corroborating Pdd1p's role in DNA elimination, we favor the hypothesis that Pdd1p structures are unique, short-lived organelles, likely to function in programmed DNA degradation and not in nucleolar biogenesis.

An abundant nucleolar phosphoprotein is associated with ribosomal DNA in Tetrahymena macronuclei.

An abundant 52-kDa phosphoprotein was identified and characterized from macronuclei of the ciliated protozoan Tetrahymena thermophila. Immunoblot analyses combined with light and electron microscopic immunocytochemistry demonstrate that this polypeptide, termed Nopp52, is enriched in the nucleoli of transcriptionally active macronuclei and missing altogether from transcriptionally inert micronuclei. The cDNA sequence encoding Nopp52 predicts a polypeptide whose amino-terminal half consists of multiple acidic/serine-rich regions alternating with basic/proline-rich regions. Multiple serines located in these acidic stretches lie within casein kinase II consensus motifs, and Nopp52 is an excellent substrate for casein kinase II in vitro. The carboxyl-terminal half of Nopp52 contains two RNA recognition motifs and an extreme carboxyl-terminal domain rich in glycine, arginine, and phenylalanine, motifs common in many RNA processing proteins. A similar combination and order of motifs is found in vertebrate nucleolin and yeast NSR1, suggesting that Nopp52 is a member of a family of related nucleolar proteins. NSR1 and nucleolin have been implicated in transcriptional regulation of rDNA and rRNA processing. Consistent with a role in ribosomal gene metabolism, rDNA and Nopp52 colocalize in situ, as well as by cross-linking and immunoprecipitation experiments, demonstrating an association between Nopp52 and rDNA in vivo.

Pdd1p, a novel chromodomain-containing protein, links heterochromatin assembly and DNA elimination in Tetrahymena.

During Tetrahymena conjugation, programmed DNA degradation occurs in two separate nuclei. Thousands of germline-specific deletion elements are removed from the genome of the developing somatic macronucleus, and the old parental macronucleus is degraded by an apoptotic mechanism. An abundant polypeptide, Pdd1p (formerly p65), localizes to both of these nuclei at the time of DNA degradation. Here we report that, in developing macronuclei, Pdd1p localizes to electron-dense, heterochromatic structures that contain germline-specific deletion elements. Pdd1p also associates with parental macronuclei during terminal stages of apoptosis. Sequencing of the PDD1 gene reveals it to be a member of the chromodomain family, suggesting a molecular link between heterochromatin assembly and programmed DNA degradation.

Identification of a novel polypeptide involved in the formation of DNA-containing vesicles during macronuclear development in Tetrahymena.

An abundant phosphoprotein with an apparent molecular mass of 65 kDa (p65) has been identified that is enriched in developing new macronuclei (or anlagen) isolated from the holotrichous ciliate, Tetrahymena thermophila. During early stages of macronuclear development, p65 is actively synthesized and deposited into young (4C) anlagen and is not found in micronuclei or parental macronuclei. p65 is not detected in older (8C) anlagen or in vegetatively growing or starved cells, and thus p65 is under stringent developmental control. In situ analyses, using polyclonal antibodies generated against p65, demonstrate that p65 undergoes a pronounced change in distribution during anlagen differentiation. Initially, anlagen are uniformly stained with these antibodies. However, following separation of conjugants, this staining pattern converts to one that is punctate and fragmented. As development proceeds, most, if not all, of these p65-based particles become peripherally located in anlagen and appear as well-defined vesicles surrounding a discrete central core of DNA of yet undetermined origin. This remarkable cytological distribution suggests an involvement of p65 in the elimination or processing of DNA during anlagen differentiation. If the above is correct, p65 provides the first inroad into the protein machinery involved in the well-known DNA rearrangements that characterize ciliate macronuclear development.