Suppression of tumor growth by intra-muscular transfer of naked DNA encoding adrenomedullin antagonist.

We have recently reported that the intra-tumoral injection of adrenomedullin (AM) antagonist (AMA; AM (22-52)) peptides significantly reduced the in vivo growth of a pancreatic cancer cell line in severely combined immunodeficient (SCID) mice. In the present study, we examined the effects of intra-tumoral and intra-muscular transfers of naked DNA encoding AMA on the in vivo growth of cancer cell lines. We demonstrate that these treatments induce the regression of a pancreatic cancer cell line and a breast cancer cell line inoculated in SCID mice. Furthermore, CD31-positive cells disappear completely from tumor tissues, following treatment, indicating that neo-vascularization is entirely inhibited. These results suggest that the intra-tumoral or intra-muscular transfer of naked DNA encoding AMA might be a promising alternative modality for treating human cancers.

A meiotic chromosomal core consisting of cohesin complex proteins recruits DNA recombination proteins and promotes synapsis in the absence of an axial element in mammalian meiotic cells.

The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.

Interferon gamma induction in human melanoma cell/allogeneic leukocyte co-cultures is enhanced by interleukin 18 but drug resistant melanoma cells are poorer inducers of IFN-gamma.

Human melanoma Colo 679 cells were made resistant to doxorubicin (adriamycin, ADM) by continuous exposure to ascending concentrations of the drug and Colo/ADM80; a variant which grew continuously in the presence of 80 ng/ml of ADM was thus established. Human peripheral blood mononuclear cells (PBMC) produced interferon gamma (IFN-gamma) when cultured with mitomycin C (MMC)-treated parental Colo 679 cells. The synthesis of IFN-gamma was synergistically enhanced by adding interleukin-18 (IL-18) and this was IL-12-dependent because a neutralizing antibody against IL-12 almost completely inhibited IFN-gamma production while control antibodies (Abs) were inactive. The cellular sources of IFN-gamma were found to be B cells, CD8+ T cells and CD4+ T cells as revealed by flow cytometry after double staining for surface antigens and staining for intracellular IFN-gamma. Interestingly, the resistant cell line induced much less IFN-gamma production than the parental cell line under the same co-culture conditions; however, IL-18 could still enhance the production of IFN-gamma. In conclusion, our study shows that acquired resistance to anti-cancer agents can also reduce immune responses to cancer cells. However, the immunostimulatory cytokine IL-18 could still enhance IFN-gamma production in drug resistant tumor cell-PBMC cultures indicating that such immunostimulatory agents could still be beneficial in immunotherapy for patients with recurrent drug resistant tumors.

Pim-1 plays a pivotal role in hypoxia-induced chemoresistance.

Hypoxia changes the responses of cancer cells to many chemotherapy agents, resulting in chemoresistance. The underlying molecular mechanism of hypoxia-induced drug resistance remains unclear. Pim-1 is a survival kinase, which phosphorylates Bad at serine 112 to antagonize drug-induced apoptosis. Here we show that hypoxia increases Pim-1 in a hypoxia-inducible factor-1alpha-independent manner. Inhibition of Pim-1 function by dominant-negative Pim-1 dramatically restores the drug sensitivity to apoptosis induced by chemotherapy under hypoxic conditions in both in vitro and in vivo tumor models. Introduction of siRNAs for Pim-1 also resensitizes cancer cells to chemotherapy drugs under hypoxic conditions, whereas forced overexpression of Pim-1 endows solid tumor cells with resistance to cisplatin, even under normoxia. Dominant-negative Pim-1 prevents a decrease in mitochondrial transmembrane potential in solid tumor cells, which is normally induced by cisplatin (CDDP), followed by the reduced activity of Caspase-3 and Caspase-9, indicating that Pim-1 participates in hypoxia-induced drug resistance through the stabilization of mitochondrial transmembrane potential. Our results demonstrate that Pim-1 is a pivotal regulator involved in hypoxia-induced chemoresistance. Targeting Pim-1 may improve the chemotherapeutic strategy for solid tumors.

Family of SRY/Sox proteins is involved in the regulation of the mouse Msh4 (MutS Homolog 4) gene expression.

The eukaryotic MutHLS-like system plays a crucial role in both mitosis and meiosis. Until now, a number of works have focused on the function of MutS and MutL homologs during mismatch DNA repair. Nevertheless, little is known about the role of these proteins during meiosis. MSH4 is a meiosis specific protein that is necessary for meiotic recombination in Saccharomyces cerevisiae. The human MSH4 protein is only found in testis and ovary. It is involved first in synapsis and second during recombination together with MLH1 (MutL Homolog 1). Here, we report the identification of the mouse Msh4 gene that is located on chromosome 3. We examined the expression of mMsh4 in testes of increasing developmental age and in elutriated germ cells. The pattern of expression during spermatogenesis is consistent with a role for MSH4 both during zygonema and pachynema. We demonstrated a promoter activity of the mMsh4 5'-flanking region by cell transfection experiments with a luciferase reporter gene. We found several SRY/Sox binding sites in this region and co-transfection experiments showed that SRY could down regulate mMsh4 promoter transcriptional activity. We propose that the regulation of mMsh4 expression could be one of the reasons for the persistence of SRY and/or SRY-related proteins in adult testis.

MSH4 acts in conjunction with MLH1 during mammalian meiosis.

MSH4 is a meiosis-specific MutS homolog. In yeast, it is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. MLH1 (MutL homolog 1) facilitates both mismatch repair and crossing over during meiosis in yeast. Germ-line mutations in the MLH1 human gene are responsible for hereditary nonpolyposis cancer, but the analysis of MLH1-deficient mice has revealed that MLH1 is also required for reciprocal recombination in mammals. Here we show that hMSH4 interacts with hMLH1. The two proteins are coimmunoprecipitated regardless of the presence of DNA or ATP, suggesting that the interaction does not require the binding of MSH4 to DNA. The domain of hMSH4 responsible for the interaction is in the amino-terminal part of the protein whereas the region that contains the ATP binding site and helix-turn-helix motif does not bind to hMLH1. Immunolocalization analysis shows that MSH4 is present at sites along the synaptonemal complex as soon as homologous chromosomes synapse. The number of MSH4 foci decreases gradually as pachynema progresses. During this transition, MLH1 foci begin to appear and colocalize with MSH4. These results suggest that MSH4 is first required for chromosome synapsis and that this MutS homologue is involved later with MLH1 in meiotic reciprocal recombination.

Cloning and expression analysis of a meiosis-specific MutS homolog: the human MSH4 gene.

The Escherichia coli MutHLS system has been highly conserved throughout evolution. The eukaryotic pathway results in a specialization of MutS homologs that have evolved to play crucial roles in both DNA mismatch repair and meiotic recombination. So far, meiosis-specific genes belonging to this family have only been identified in yeast. In Saccharomyces cerevisiae, MSH4 (MutS homolog 4) is a meiosis-specific protein that is not involved in mismatch correction. This protein is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. We have identified the human MSH4 homolog gene. The predicted amino acid sequence shows 28.7% identity with the S. cerevisiae MSH4 protein. By Northern blot analysis, human MSH4 transcripts are only detectable in testis and in ovary with a lower level of expression. We have mapped MSH4 to human chromosome 1 at band p31 by fluorescence in situ hybridization. The identification of such a gene provides a powerful tool for clarifying the respective roles of MSH and MLH genes in mammalian meiosis.