Identification of drm, a novel gene whose expression is suppressed in transformed cells and which can inhibit growth of normal but not transformed cells in culture.

Using differential display analysis, we compared the expression of RNA in v-mos-transformed cells and their flat revertant and isolated a novel gene, drm (down-regulated in mos-transformed cells), whose expression is down-regulated in parental v-mos-transformed cells but which is expressed at a high level in the revertant and normal rat fibroblasts (REF-1 cells). Analysis of different oncogene-transformed cells revealed that drm gene expression was also suppressed in REF-1 cells transformed by v-ras, v-src, v-raf, and v-fos. The drm cDNA contains a 184-amino-acid-protein-encoding open reading frame which shows no significant homologies to known genes in DNA databases. Polyclonal antibodies raised against drm peptide detect a protein with the predicted size of 20.7 kDa in normal cells and under nonpermissive conditions in cells conditionally transformed by v-mos but not in parental v-mos-transformed cells. Northern analysis of normal adult tissues shows that drm is expressed as a 4.4-kb message in a tissue-specific manner, with high expression in the brain, spleen, kidney, and testis and little or no expression in the heart, liver, and skeletal muscle. In situ hybridization analysis in adult rat tissue reveals good correlation with this pattern and indicates that drm mRNA is most highly expressed in nondividing and terminally differentiated cells, such as neurons, type 1 lung cells, and goblet cells. Transfection of a drug-selectable drm expression vector dramatically reduced the efficiency of colony formation in REF-1 and CHO cells, and the drm-transfected REF-1 survivors expressed low or nondetectable levels of exogenous drm mRNA. The toxic effects of drm can be overcome by cotransfection with constructs expressing oncogenic ras; furthermore, cells expressing high levels of drm and conditionally transformed with mos-expressing Moloney murine sarcoma virus rapidly undergo apoptosis when shifted to the nonpermissive temperature. Taken together, our data suggest that cells expressing high levels of drm undergo apoptotic death in the absence of oncogene-induced transformation and that drm represents a novel gene with potential roles in cell growth control or viability and tissue-specific differentiation.

Localization of alpha 2A- and alpha 2B-adrenergic receptor subtypes in brain.

Recent studies have shown that all three subtypes of alpha2-adrenergic receptor (alpha2-AR) are found in brain. The purpose of this study was to map the subtype localization of the alpha2A- and alpha2B-ARs in brain structures. RNase protection shows that both the alpha2A- and alpha2B-ARs are detectable in cortex, cerebellum, pons-medulla, and hypothalamus. We tested probes derived from the alpha2A- and alpha2B-AR cDNAs on cell lines that express each of the alpha2-AR subtypes to establish the subtype specificity of these probes for in situ hybridization. Then we used the alpha2A- and alpha2B-AR probes for in situ hybridization on sagittal and coronal sections of rat brain. Both alpha2A and alpha2B mRNA were detected throughout the brain. Overall, there appears to be a greater expression of message for alpha2A- than alpha2B-AR in most brain areas, with the exception of the thalamus. Developing these probes for in situ hybridization is an important step for further studies on the exact role of the alpha2-AR subtypes in neurons that modulate cardiovascular function.

Diverse tissue expression of rat alpha 2-adrenergic receptor genes.

Previously, we have reported two major alpha 2-adrenergic receptor transcripts in rat brain of 3.8 and 3.0 kb and the cloning and characterization of the rat brain complementary DNA (cDNA) (RB alpha 2C) specific for the 3.0-kb messenger RNA. In this report, we used rat brain cDNAs specific for the 3.0 and 3.8 kb transcripts, which encode the alpha 2C- and alpha 2A-adrenergic receptors, respectively, and the RNG alpha 2 cDNA, which encodes for the nonglycosylated alpha 2B-adrenergic receptor in rat, to study tissue-specific expression of the three alpha 2-adrenergic receptor genes in rat. To eliminate cross-hybridization of probes with transcripts from other alpha 2 genes, we subcloned fragments that encode for the highly divergent third cytoplasmic loop of each rat alpha 2-adrenergic receptor cDNA and used RNase protection analysis to detect specific transcripts. We show that the three rat alpha 2-adrenergic receptor genes have diverse patterns of tissue expression, and although transcripts specific for each alpha 2-adrenergic receptor gene are found in brain and kidney, the levels of expression of each subtype differ in these tissues. We speculate on the significance of tissue-specific expression of the alpha 2-adrenergic receptor genes.

[Specific interaction of a nuclear protein factor from the CTF/NF-I family with 2 different promoter regions of the rat tyrosine aminotransferase gene]. / Spetsificheskoe vzaimodeistvie iadernogo belkovogo faktora semeistva CTF/NF-I s dvumia raznymi uchastkami promotora gena tirozinaminotransferazy krysy.

Using gel-retardation and DNase I footprinting assays, we have analysed sequence-specific DNA-protein interactions within proximal promoter fragment (from -2 to -210 bp relative to the transcription start) of the rat tyrosine aminotransferase (TAT) gene. Two distinct DNase I protection regions flanked at either boundary by sites of DNase I hypersensitivity were observed with the rat-liver nuclear extracts. The internal sequence of the region I non-coding strand, (-155)TGGGCCACCTTCCAAT(-170), is highly homologous to the NF-I consensus sequence TGG(N)6-7TGCCAA and also shares a CCAAT-box; the region II noncoding strand sequence includes asymmetrically positioned (-37)AGCCAAT(-43) recognition motif. Since there have been a number of reports about multiple DNA-binding factors that recognize CCAAT homologies, both regions were likely to interact with either a single or distinct factors. Here we show that both region I and II of the TAT gene promoter are binding to the same factor related to the human CTF/NF-I. The evidence for that is based on competition experiments using the DNA fragment containing a synthetic consensus sequence for the NF-I recognition site and on the indistinguishable chromatographic properties of the activity specifically binding to each of three DNA fragments containing NF-I consensus, region I and region II sequences.

[Inhibition of proliferation of normal and neoplastic liver cells under conditions of prolonged hormonal stimulation]. / Tormozhenie proliferatsii normal'nykh i opukholevykh kletok pecheni v usloviiakh dlitel'noi gormonal'noi stimuliatsii.

Single injections of rats with hydrocortisone led to the inhibition of regenerating liver cell proliferation and protooncogene++ Ha-ras mRNA synthesis within 48 hours of hormonal induction. Administration of hydrocortisone to rats daily for 10 days resulted in a persistent decrease of the liver cell capacity to proliferate in response to partial hepatectomy. This inhibiting effect was observed for at least 7 days after cessation of hormonal stimulation; the level of Ha-ras mRNA was thereby decreased. A marked inhibition of ascite hepatoma cell growth was demonstrated after injections of those cells to mice induced with hydrocortisone for 10 days. Such a persistent effect of hydrocortisone is thought to be due to the depletion of the hormone-dependent hepatotrophic factors. The effect of the glucocorticoid hormone in vivo can be supposed to involve both the direct and indirect regulation of target cell proliferation. The latter is mediated via the changes in the activity of exogenous factors which control cell growth and proliferation.

[Effect of glucocorticoid hormones on DNA synthesis and expression of ras proto-oncogenes in proliferating rat liver cells]. / Deistvie gliukokortikoidnykh gormonov na sintez DNK i ékspressiiu protoonkogena tipa ras v kletkakh proliferiruiushchei pecheni krys.

A single injection of partially hepatectomized rats with glucocorticoids results in the blocking of DNA synthesis as well as in the inhibition of the protooncogene Ha-ras-1 mRNA accumulation in proliferating rat liver cells. The kinetics of the both hormone-induced effects differ from those observed for tyrosine aminotransferase induction. The effect of glucocorticoids persists for at least 48 hours and does not depend on the time of the hormone injection.

[Biochemical and functional characteristics of thymus and spleen lymphocytes in C3HA mice during the growth of hepatoma 22 and after immunization with sheep erythrocytes]. / Biokhimicheskaia i funktsional'naia kharakteristika limfotsitov timusa i selezenki myshei C3HA pri roste gepatomy 22 i posle immunizatsii éritrotsitami barana.

Activities of key enzymes of purine metabolism [adenosine deaminase (AD); purine nucleoside phosphorylase (PNP); 5'-nucleotidase] were studied; changes in DNA content, nucleus ploidity in thymocytes, T- and B-lymphocytes in the C3HA mouse spleen during solid 22 hepatoma growth and after the immunization were monitored. Immunological properties of lymphocytes were also investigated measuring antibody formation and the reaction of blasttransformation in response to phytohemagglutinin, concanavalin A and lipopolysaccharide. Within the first 48 hrs after the tumor implantation and immunization certain nonspecific biochemical mechanisms of lymphocytes activation (elevated AD activity, decreased activity of 5'-nucleotidase, augmented intracellular DNA levels, polyploidity) were revealed. As the solid 22 hepatoma reached the maximum growth rate specific alterations in the activities of the purine metabolism key enzymes were observed reflecting the response of thymus and spleen lymphocytes to the presence of the malignant tumor.