(Proto-myb) in Hemopoietic Tissues - Molecular and Cellular Biology

MOLECULAR AND CELLULAR BIOLOGY, JUlY 1984, p. 1206-1212 0270-7306/84/071206-07$02.00/0 Copyright © 1984, American Society for Microbiology

Vol. 4, No. 7

Expression of a Proto-Oncogene (Proto-myb) in Hemopoietic Tissues of Mice DIANA SHEINESS* AND MINNETTA GARDINIER Department of Biochemistry, Louisiana State University Medical School, New Orleans, Louisiana 70112 Received 28 February 1984/Accepted 10 April 1984

This study addressed the possibility that proto-myb (also called c-myb), the cellular homolog of a retroviral transforming gene, plays a role in hemopoiesis, particularly during maturation of T cells. By gel blot hybridization, we confirmed previous reports that proto-myb transcripts are found at much higher levels in thymic lymphocytes and cells of the erythroid lineage than in other tissue sources. Using dot blot hybridizations, we demonstrated further that similar levels of proto-myb expression are found in thymic lymphocytes taken from young mice with active thymuses and from old mice whose thymuses have undergone involution and that the extent of proto-myb expression decreases at least 10-fold as T cells progress from immature cortical thymocytes to the mature, resting T cells taken from lymph nodes. These results suggest that the protein product of proto-myb functions during T-cell differentiation.

In recent years, evidence has accumulated that indicates a host origin for at least 16 different retroviral oncogenes (3). By a recombination mechanism not fully understood, the viral genomes have evidently transduced these otherwise normal and evolutionarily conserved cellular genes (protooncogenes) (8). Once such a recombinant virus infects a susceptible target cell, malignant growth results by expression of either abnormally high amounts of the oncogene protein or an altered form of the protein (3). Furthermore, numerous examples of aberrant expression of proto-oncogenes have been found in transformed cell lines and in tumors whose induction apparently did not involve retroviral oncogenes (35). The evidence is compelling that many cancers result from abnormal expression of a proto-oncogene. The description of a proto-oncogene is strictly operational; membership in this class of cellular genes requires only that there be strong homology between the proto-onc and a retroviral oncogene (v-onc). It is still not known exactly how any of the viral oncogene proteins induce malignant growth. One closely related family of viral oncogenes, including vsrc, v-fps, v-abl, v-yes, and v-ros, encodes membrane-bound proteins with tyrosine-specific protein kinase activity (3). However, the means by which these kinase activities cause transformation remains a mystery. Two other viral oncogenes, v-myc and v-myb, produce proteins that are located in the nucleus of transformed cells, but these have not been assigned any enzymatic activity (1, 10; K.-H. Klempnauer and J. M. Bishop, personal communication). Nonetheless, the similarity between viral genes with known tumorigenic activity and the proto-oncogenes has led to the hypothesis that proto-oncogenes, under normal circumstances, may function in the regulation of cell growth or differentiation or both. In support of this hypothesis, recent work has suggested that proto-fos and proto-fms may play a role in embryonal or extraembryonal tissues during development (22-25). The theory that proto-oncogenes participate in the control of cell division has been corroborated most dramatically by the finding that v-sis, the oncogene of the simian sarcoma virus, encodes a protein that is similar or identical to the plateletderived growth factor (11, 38) and that v-erbB, the oncogene of avian erythroblastosis virus, closely resembles the epider*

mal growth factor receptor (12). These remarkable findings for sis and erbB begin at last to close the gap between physical and functional studies of viral and cellular oncogenes. In this study, expression of proto-myb in hemopoietic tissues in mice was investigated, with a particular focus on learning whether this gene might function during the differentiation of T cells or involution of the thymus. Previous studies with chicken tissues and with permanent cell lines have indicated that proto-myb expression, in contrast to that of other proto-onc genes, is confined to only hemopoietic tissues (7, 9, 16, 39). To trace the physiological function of proto-myb, we have expanded previous studies of proto-myb expression as follows. The relative amounts of RNA transcribed from proto-myb were measured in several hemopoietic organs of known cellular composition, including fetal liver, bone marrow, spleen, thymus, and mesenteric lymph nodes. In addition, immature T cells in the thymic cortex were separated from the more mature medullary thymocytes, and proto-myb expression was measured in each fraction. Lastly, the expression of proto-myb was measured as a function of the naturally occurring age-dependent regression of the thymus. MATERIALS AND METHODS Animals. Cells and tissues were obtained from inbred BALB/c ByJ mice; our mouse colony was derived from pedigreed breeding pairs that were provided originally by The Jackson Laboratory, Bar Harbor, Maine. Preparation of RNA. For most of the RNA preparations, whole organs were minced and added to ca. 10 ml (per organ) of homogenization buffer containing 0.1 M NaCl, 10 mM Tris (pH 7.3), 5 mM EDTA, and 200 p.g of proteinase K (EM Biochemicals) per ml. For the thymus, spleen, and mesenteric lymph node preparations, the minced organs were forced through a 40-mesh stainless steel screen which retained connective tissue and allowed only hemopoietic cells to pass through; these cells were washed in phosphatebuffered saline (PBS; 0.02 M NaH2PO4, 0.08 M NaHPO4, 0.15 M NaCl) and suspended in homogenization buffer. For thymus, this procedure effectively removed dividing cells from our preparations. Only ca. 1% of cells in the thymus are actively undergoing division, and most of these are tightly

Corresponding author. 1206

EXPRESSION OF PROTO-mvb IN MICE

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FIG. 1. Transcription of proto-myb and a gene for a ribosomal protein in several mouse tissues. Polyadenylated RNA was fractionated on 1% agarose-formaldehyde gels, transferred to nitrocellulose, and then hybridized to 32P-labeled DNA as indicated. (A and B) Hybridization to labeled v-myb DNA; (C) hybridization to labeled plasmid L7 DNA. (A) 4 ~.ig of RNA was applied to each lane; (B and C) 3.5 and 1 p.g of each RNA was loaded onto adjacent lanes.

thymic epithelium; thus, such cells were along with the connective tissue. Bone marrow was obtained from tibias and femurs by removal of the epiphyses and injection of PBS to wash out the cells. Cytocentrifuge slides were prepared from bone marrow cells, and cell types were identified by microscopic examination with the assistance of Albert Romanosky. Cells were lysed by the addition of sodium dodecyl sulfate to 1% just before homogenization with a Polytron tissue homogenizer. Homogenates were digested with proteinase K for 1 h affiliated with the retained

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proteinase K-digested homogephenol-chloroform and precipitatml of 10 ed with ethanol. Pellets were suspended in 1 to mM Tris (pH 7.3)-7 MM MgCl2 and digested with 15 ~tg of For total cellular RNA,

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samples were extracted once with phenol-chloroform and then precipitated with ethanol. For preparation of polyadenylated RNA, we followed the previously published procedure (16) described here in brief. Proteinase K-digested homogenates were stirred with 1 to 3 ml (packed volume) of oligodeoxythymidylic acid-cellulose in 0.5 M NaCl for 1 h, and then the oligodeoxythymidylic acid-cellulose was collected by centrifugation and transferred to a column. The column was washed, and polyadenylated RNA was eluted as described previously (16) and then precipitated with ethanol. Polyadenylated RNA was treated with DNase I

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Analysis on ethidium bromide-stained formaldehyde-agarose gels (see below) indicated that these RNAs were virtually free of contamination with rRNA. Analysis of RNA on formaldehyde-agarose gels. As previously described (16), samples were applied to slab gels containing 1% agarose and subjected to electrophoresis in buffer containing 2.2 M formaldehyde to ensure denaturation of the RNA samples (19). Gels were soaked to remove formaldehyde, stained for 15 min with 2 p.g of ethidium

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bromide per ml, destained for 8 h or longer, and photographed through a Wratten no. 22 filter. For hybridization, polyadenylated RNA was transferred to Schleicher & Schuell nitrocellulose (34), baked at 80°C under vacuum for 2 h, and then hybridized to 32P-labeled DNA as described below. Dot blot analysis of RNA. As in the method of Thomas (34), samples of total cellular RNA were serially diluted, heated to 75°C, and then applied in 20 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) to nitrocellulose sheets with a Schleicher & Schuell Minifold apparatus. For each RNA sample, absorbance at 260 nm was read just before serial dilution. For the determination of relative RNA levels, nicked plasmid DNAs were also serially diluted and applied to each filter; plasmid DNAs were boiled and quick-chilled before application to filters. Standards present on each filter were pL7 (kindly provided by R. Perry), a plasmid whose cloned insert corresponds to mRNA for one of the ribosomal proteins (21), and plasmid pVM2 (provided by T. Gonda and J. M. Bishop), which contains the v-myb gene of avian myeloblastosis virus. Preparation and hybridization of 32P-labeled cDNAs. DNAs were radiolabeled by nick translation (30) in the presence of [a-32P]dATP (800 Ci/mmol; New England Nuclear Corp., Boston, Mass.). Templates for labeling were plasmid pL7 (described above) and a 1-kilobase fragment of DNA derived from plasmid pVM2 by digestion with restriction endonucleases KpnI and SacI; this fragment contains nearly the entire v-myb gene (33). Dot blot and RNA gel blot filters were hybridized for 3 days to ca. 107 cpm of denatured 32P-labeled DNA per filter. Annealing of plasmid pL7 probe was in buffer containing 50% formamide as previously described (16); for hybridization of plasmid pVM2 probe, the formamide content of the buffer was reduced to 30%o to permit formation of partially mismatched hybrids. After hybridization, filters were washed for 2 to 3 h with 0.1 x SSC-0.1% sodium dodecyl sulfate at 53°C for plasmid pL7 hybridizations and 0.5 x SSC-0.1% sodium dodecyl sulfate at 37°C for plasmid pVM2 hybridizations. Dried filters were exposed to Kodak XRP-5 X-ray film for 1 to 14 days in the presence of Dupont Cronex Lightning-Plus intensifying screens. Fractionation of thymic lymphocytes. Suspensions of freshly prepared thymocytes were found to be >95% viable according to trypan blue exclusion and >95% positive for expression of Thy 1 antigen according to a standard cytotoxicity assay (6). Monoclonal anti-Thy 1 was obtained from Miles-Yeda Ltd., Rehovot, Israel. Fractionation with peanut agglutinin (PNA; EoY Laboratories, Inc., San Mateo, Calif.) was carried (5) out by one of two methods. For agglutination in suspension, thymocytes were adjusted to 107 cells per ml in PBS containing 1 mM MgSO4 and 2 mM CaC12 (PBS-MgCa). Suspended cells were mixed with an equal volume of 1 mg of PNA per ml in PBS. Three milliliters of this mixture was layered onto a 7-ml cushion of 50% fetal calf serum in PBS-MgCa; clumped cells were allowed to settle for 20 min and were then collected from the bottom of the tube. Nonagglutinated cells were aspirated from the top of the cushion after an additional 10 min. Of the starting number of cells, 65% were recovered after this procedure, and about 80% of the recovered cells were found in the PNA-positive fraction. Cells were washed with PBS-MgCa containing 0.2 M galactose to remove PNA and then used for RNA extraction as described above. For the second method, we employed the panning technique (20) as modified for use with PNA by E. Rothenberg (personal communication). Petri dishes were coated with PNA and

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SHEINESS AND GARDINIER

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