Tissue-Specific Expression of mRNAin Mouse Lymphocytes Detected by v-fos but not by Human c-fos DNAProbes

Transcription of the c-fos gene is known to be induced transiently by many types of cellular stimuli in various cultured cell lines; however, several authors have reported that the c-fos gene is constitutively transcribed in lymphoid cells. We detected, in fact, abundant transcripts which hybridized with a \-fos DNA probe in nuclear run-off transcripts and poly(A)+ RNAof both quiescent mouse splenic lymphocytes and unstimulated monocytic tumor cell lines. However, humanc-fos CDNAdid not hybridize with most of these transcripts. No signal was detected by \-fos probe in nuclear run-off transcripts of 3T3 fibroblasts, and c-fos was inducible in both the 3T3 cells and the monocytic tumor cell lines. In quiescent lymphocytes, only the 0.3 kb HincII-Pvull portion of y-fos DNA,which contains a repeat of CAAAA, hybridized with these transcripts; neither other parts of \-fos nor humanc-fos DNAsdid. These results suggest that a significant portion of the previously reported 'constitutive' transcripts detected by y-fos DNAin lymphocytes and monocytes are not transcripts of c-fos but of other sequences which are specifically expressed in lymphoid cells and have homology with the 0.3 kb Hincll-Pvull fragment of y-fos. The proto-oncogene c-fos is one of the immediate early genes (1) whose expressions are induced by various stimulations that lead cells to either proliferation or differentiation (2, for review). After stimulation, transcription of the c-fos gene is activated within minutes without new protein synthesis, suggesting that preexisting protein(s) are posttranslationally modified to activate the transcription of this gene (3). Several DNAelements which are required for induction and trans-acting factors that bind to these sequences have been identified (reviewed in Refs. 4 and 5). However, it is yet to be determined whether the induction mechanisms are common amongcell types or are cell type specific. In 3T3 fibroblasts, A431 epithelial cells, and PC12 pheochromocytoma cells, the induction occurs at the transcriptional level (3, 6, 7), whereas constitutive transcription of the c-fos gene has been observed in monocytes and Band T-lymphocytes (8, 9, 10). In these cells, the molecular mechanismsof constitutive transcription and posttranscriptional regulation of the c-fos gene are as yet unclear. Webegan with a nuclear run-off assay of mouse nonadherent lymphocytes to determine the contribution of the transcriptional regulation to the induction of c-fos mRNA. Weconfirmed that constitutive transcripts were detected by a w-fos probe in unstimulated lymphocytes. Detailed analysis of those transcripts showed that these Abbreviations used: Con A, concanavalin A; TPA, 12-O-tetradecanoyl-phorbol 13 acetate. were detected only when a part of v-fos DNAwas used as a probe: they were detected neither by other parts of v-fos DNAnor by human c-fos CDNA.Also, in monocytic cell lines v-fos DNAdetected constitutive run-off transcripts, whereas human c-fos DNAdetected inducible ones. In Northern analysis of poly(A)+ RNAfrom lymphoid cells, the 1.9 kb mRNAthat is constitutively expressed in commonamong them was detected by the v-fos DNAprobe in addition to inducible 2.2 kb c-fos mRNA. MATERIALS AND METHODS Cell culture. Lymphocyteswere prepared from spleens of CD1 (ICR) mice using Ficoll-Hypaque gradient separation. After adherent cells were removed by adhesion to a plastic dish, non-adherent lymphocytes were incubated for 6 to 12 h before initiating the experiments in RPMI1640 medium with 10% fetal calf serum (FCS). Balb/c 3T3 and NIH 3T3 fibroblasts (provided by the Japanese Cancer Research Resources Bank) were maintained in MEMwith 10% FCS and DMEM with 10% calf serum, respectively. Con fluent and serum starved cultures were used. Mouse macrophage cell line P388D1 and myelomonocytic cell line WEHI3(kindly provided by Prof. K. Kumagai, Tohoku University) were maintained in RPMI1640 supplemented with 10% FCS and used at the exponentially growing phase. Hybridizations. A common buffer was used in hybridizations of nuclear run-off transcripts and Northern and Southern blots to probes. The buffer, based on the description