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Expression of a Mouse Long Terminal Repeat Is Cell Cycle-Linked (Friend Cells/Gene Expression/Retroviruses/Transformation/Onc Genes) LEONARD H

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Expression of a Mouse Long Terminal Repeat Is Cell Cycle-Linked (Friend Cells/Gene Expression/Retroviruses/Transformation/Onc Genes) LEONARD H Proc. Natl. Acad. Sci. USA Vol. 82, pp. 1946-1949, April 1985 Biochemistry Expression of a mouse long terminal repeat is cell cycle-linked (Friend cells/gene expression/retroviruses/transformation/onc genes) LEONARD H. AUGENLICHT AND HEIDI HALSEY Department of Oncology, Montefiore Medical Center, and Department of Medicine, Albert Einstein College of Medicine, 111 East 210th St., Bronx, NY 10467 Communicated by Harry Eagle, November 26, 1984 ABSTRACT The expression of the long terminal repeat have regions of homology with a Syrian hamster repetitive (LTR) of intracisternal A particle retroviral sequences which sequence whose expression is also linked to the G, phase of are endogenous to the mouse genome has been shown to be the cell cycle (28). linked to the early G6 phase of the cell cycle in Friend erythroleukemia cells synchronized by density arrest and also MATERIALS AND METHODS in logarithmically growing cells fractionated into cell-cycle Cells. Friend erythroleukemia cells, strain DS-19, were compartments by centrifugal elutriation. Regions of homology grown in minimal essential medium containing 10% fetal calf were found in comparing the LTR sequence to a repetitive serum (28). Cell number was determined by counting an Syrian hamster sequence specifically expressed in early G1 in aliquot in an automated particle counter (Coulter Electron- hamster cells. ics). The cells were fractionated into cell-cycle compart- ments by centrifugal elutriation with a Beckman elutriator The long terminal repeats (LTR) of retroviral genomes rotor as described (29). For analysis of DNA content per contain sequence elements that regulate the transcription of cell, the cells were stained with either 4',6-diamidino-2- the viral genes (1). The enhancer and promoter elements of phenylindole (Fig. 1; ref. 30) or propidium iodide (Fig. 4 these LTRs can also influence the expression of cellular Left; ref. 29), and the DNA content per cell was measured by genes at sites near which they integrate or to which they are flow cytometry. transposed (2). This was first demonstrated in the case of Plasmids and hybridization. Cytoplasmic dot blots were chicken lymphomas, in which the LTR of the avian leukosis prepared by the method of White and Bancroft (31) as virus (ALV) genome is inserted in the host genome near the described (20). Plasmids were labeled with [32P]dCTP (New proto-onc gene c-myc, thereby activating c-myc expression England Nuclear, 3000 Ci/mmol; 1 Ci = 37 GBq) by nick- (3-5). Similar results have been reported for the activation of translation (32). The plasmids used were pMCT-1, which c-erbB by the ALV genome in chicken erythroblastosis (6) contains a 605-base-pair (bp) cDNA clone of an IAP LTR and the intl and int2 loci in mouse mammary tumors induced (20), and pCR1, a cDNA clone of mouse p-globin (33). by mouse mammary tumor virus (MMTV) (7-9). Vertebrate genomes also carry a large number of endog- RESULTS enous retroviral sequences that contain flanking LTRs. In Friend erythroleukemia cells, strain DS-19, contain a high the mouse, IAP (intracistemal A particle) sequences are a level of transcripts of endogenous IAP genomes (20). When family of endogenous retroviral sequences whose transcripts these cells are seeded at a density of 1 x 105 cells per ml, are packaged in A particles in the cisternae of the they grow rapidly, increasing in number by about 20-fold in endoplasmic reticulum (10-12). A particles and TAP gene 4 days, after which they cease dividing, and growth is expression are found in early mouse embryogenesis (13-16). arrested in the G1 phase of the cell cycle (34). This was Reappearance of the particles and presence of UAP tran- confirmed by the data of Fig. 1 in which 65% of such arrested scripts is found only in transformed cells in a variety of DS-19 cells (0 time, which is 4 days after seeding) have a 2N, mouse tumors, including myelomas (17), leukemias (18-20), or G1, content of DNA, as measured by flow cytometry. and a dimethylhydrazine-induced mouse colon tumor When diluted into fresh, prewarmed medium, the cells were (20-22). The significance of this consistent reexpression of released from this block, and within 24 hr, most of the cells retroviral sequences in transformation is not known. We were cycling, with 78% having the DNA contents of S or have reported, however, that an UAP LTR contains a region G2/M cells (Fig. 1). By day 4, the cells again became homologous to a core sequence found in transcriptional arrested in G1, where they remained if not refed. enhancer elements (20), and in two myelomas an IAP LTR The level of cytoplasmic RNA that hybridized to pMCT-1, has been transposed into c-mos, thereby activating its tran- a cDNA probe of an IAP LTR (20), was markedly decreased scription (23-26). Presumptive evidence for down-stream during the 24 hr after dilution of the cells (Inset of Fig. 2 promotion by other endogenous retroviral LTRs in murine Upper). Fig. 2 Upper presents the average of the quantita- cell transformation also has been published (27). tion of such dot blots from a number of experiments. Within In situ hybridization experiments of an TAP LTR sequence 6 hr of reseeding, the level of IAP LTR transcripts dropped to frozen sections of a mouse colon tumor showed that by >90%. The level remained relatively low over the next 3 highest expression was often found in cells lying side by days during logarithmic growth, as indicated by the rapid side. This suggested to us that expression of these sequences increase in the number of cells per ml, also shown in Fig. 2 may be linked to the G, phase of the cell cycle and that the Upper. When the cells became arrested again, beginning on doublets represented daughter cells having recently divided day 3, the level of hybridization rose and continued to (22). This question has now been studied in Friend increase as the cells remained stationary. Fig. 2 Lower erythroleukemia cells. The data demonstrate that expression shows similar data on RNA levels at early time points after is indeed elevated in G1. Further, the TAP LTR was found to reseeding. Hybridization stayed relatively constant for up to The publication costs of this article were defrayed in part by page charge Abbreviations: LTR, long terminal repeat; IAP, intracisternal A payment. This article must therefore be hereby marked "advertisement" particle; MMTV, mouse mammary tumor virus; Me2SO, dimethyl in accordance with 18 U.S.C. §1734 solely to indicate this fact. sulfoxide. 1946 Biochemistry: Augenficht and Halsey Proc. Natl. Acad. Sci. USA 82 (1985) 1947 GI G2/M 0 3 6 12 24 IS I E a) 0 3hr 6hr 12 hr 60- I0 x G) It U) :3 20 ._~~~~~~~~~~~~~~~~~~J 0) 2 3 Mf- Cc) of" .2- / - . Timeda 0 60- 24hr 2 day 5day 6day 20- 20 60 100 140 180 Time, min FIG. 2. Expression of TAP LTR sequences. DS-19 cells were grown as in Fig. 1. At various times after reseeding (days in Upper; minutes in Lower), the cell concentration was determined using a Coulter Counter, and dot blots of the cytoplasmic fraction were made by the method of White and Bancroft (31). Aliquots of the FIG. 1. Cell-cycle progression of DS-19 cells. DS-19 cells were cytoplasm for each time point, representing cytoplasmic RNA from grown in minimal Eagle's medium containing 10% fetal calf serum to a constant cell number (10-10, depending on the experiment) were a density of 2-3 x 106 cells per ml ("0" time) and then reseeded into spotted on nitrocellulose and hybridized to an 1AP LTR cDNA fresh prewarmed medium at 1 x lot cells per ml. Aliquots of cells clone, pMCT-1, labeled with 32P by nick-translation. (Upper Inset) were taken at 3, 6, 12, and 24 hr and at 2 days, 5 days, and 6 days Results of a typical experiment (time in hours after reseeding). The after reseeding; the DNA was stained with 4',6-diamidino-2- results from each experiment were quantitated by scanning and phenylindole, and the DNA content per cell was analyzed by flow integrating the peak areas with a Quick Scan Jr. densitometer cytometry. 10,000 or more cells were analyzed in the G1 peak. (Helena Laboratories, Beaumont, TX). For each experiment, the 0 time point was normalized to 100%, and the results for the other time points were calculated in relation to this value. Each point 90 min but decreased abruptly to 20% of the 0 time level by shown in the figure represents the mean of at least two independent 3 hr. Since most cells still had a G1 DNA content at 3 hr (Fig. experiments with different cell cultures, preparations, and probes. 1), this indicates that the high level ofexpression is restricted Also shown in Upper is the growth curve (A) in cells per ml from a to the early G, portion of the cycle. typical experiment. Fig. 3 presents the results of an experiment in which IAP LTR levels were quantitated by dot-blot analysis in cells markedly from the pattern of accumulation of the IAP LTR diluted into fresh medium or induced to differentiate by sequences. Similarly, we found that the content of cytoplas- adding 1.5% dimethyl sulfoxide (Me2SO) to the fresh me- mic rRNA increases rapidly as the cells enter the cell cycle dium. Again, hybridization was greatly reduced within 3 hr (not shown). In two other reports in which sequences related in the presence as well as absence of the inducer. In to growth were identified in density-arrested cell populations addition, reaccumulation of IAP LTR transcripts in the released by refeeding with fresh serum, <1% of cDNA induced culture lagged behind that of the uninduced culture A by 1 day (5 days versus 4 days).
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