Proc. Natl. Acad. Sci. USA Vol. 82, pp. 1946-1949, April 1985 Biochemistry Expression of a mouse is cell cycle-linked (Friend cells/ expression/retroviruses/transformation/onc ) 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 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.

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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). This is because the induced 200- I cell population became arrested in G, later than the If uninduced population (not shown), which may be related to . the transient cell-cycle retardation that occurs early after 4 exposure of the cells to inducer (29, 34). This transient .I- *300 C retardation in G1 in induced cells between 12 and 20 hr does C 100' 8 not result in any apparent increase in IAP LTR expression. 8 O This may be due to several factors. First, the synchrony at 200 this time after release may not be great enough to see an voo 50- increase. Second, because the increase in LTR expression is 100 o in the early part of G1, the retardation may take place in a later portion of G1. Third, it is likely that cells that are retarded in the cell cycle with a G, DNA content and that are 0.5 1 2 3 4 5 6 undergoing differentiation are biochemically distinct from Time, days those progressing through G1 (hence the retardation) or those arrested until refeeding in therefore, this G1 arrest FIG. 3. Expression of IAP LTR sequences in induced and GI; uninduced cells. DS-19 cells were reseeded into fresh prewarmed may simply not involve high levels of IAP LTR expression. medium (o) or medium containing 1.5% Me2SO (A), and cell aliquots Fig. 3 also shows that the content of globin mRNA in the taken at times thereafter were analyzed for the level of cytoplasmic cells induced by fresh medium containing Me2SO began to RNA that hybridizes to pMCT-1 as described in Fig. 2. The content increase at day 2 and peaks at day 3, declining thereafter. of globin mRNA also was assayed in the induced cells (v) by This is consistent with published work on the induction of hybridization of a duplicate blot to a nick-translated clone of a globin gene expression in Friend cells (35) and differs mouse A3-globin cDNA. 1948 Biochemistry: Augenlicht and Halsey Proc. Natl. Acad. Sci. USA 82 (1985) GI G2/m f +

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1 2 3 4 5 6 7 8 Fraction FIG. 4. Expression of 1AP LTR sequences in cell-cycle compartments. DS-19 cells in midlogarithmic growth (2 days after refeeding) were fractionated by centrifugal elutriation. Aliquots of each cell fraction were stained with propidium iodide, and the DNA per cell was quantitated by flow cytometry. (Left) Flow cytometry data for cells of each fraction. (Right) Hybridization data to pMCT-1 for each fraction, as assayed in Fig. 2. clones screened exhibited differences in the level of expres- sequences are essentially random selections from highly sion between the arrested state and the growing state (28, repetitive sequence families, we believe the homology is of 36). These data all indicate that the changes in level of interest. Although speculative, these data may indicate that expression of the IAP LTR sequences are not generally seen there are related repetitive sequence elements in different for other RNA molecules. species linked to genes associated with cell-cycle progres- The IAP LTR has no significant homology to other retro- sion. viral LTRs, and we found no hybridization of the IAP LTR clone pMCT-1 (20) to a cloned sequence ("CHF"; A. Oliff, DISCUSSION personal communication) that is representative of the LTRs We have presented evidence that the level of IAP LTR of the two Friend virus complex components, spleen focus- transcripts in Friend erythroleukemia cells is highest in the forming virus and Friend murine leukemia virus (not shown). G1 phase of the cell cycle. In this report, we refer to the This makes it unlikely that the results seen are due to such arrested cells as G1 cells, rather than Go, for two reasons. cross-hybridization. First, they have a G1 DNA content; second, the content of In order to determine if expression of the IAP LTRs is also IAP LTR transcripts is elevated in both the arrested cells highest in cycling cells as they pass through G1, we fraction- and in cycling cells in G1. It is possible, however, that there ated log DS-19 cells into cell-cycle compartments by cen- are other important biochemical differences between the trifugal elutriation. Fig. 4 Left shows the flow cytometry cycling G1 cells and the arrested cells, indicating that the data indicating that fraction 1 contained almost a pure G1 latter should be considered Go cells, but this issue is not population of cells, and subsequent fractions contained cells addressed by our data. in G1, S, and G2. Fig. 4 Right shows the relative hybridiza- 529 tion of an TAP LTR probe to dot blots of the fractions in Fig. pMCT-1 TCCTTAAGAGGGACGGGGTTTTCGTTTTCTCTCTCTTGCTCT 4 Left. The data show that highest levels of expression were P2A8 TCCT------GACGGGGTT*T*GT*TTCTCT*TCTTGCTCT indeed associated with G1. These experiments demonstrate that the elevation in expression of IAP LTR sequences is T GATTGAAG A AAA characteristic of G1 cells, whether obtained from a stationary-cell population by density inhibition or from a cycling population by elutriation. 445 Finally, Hirschhorn et al. (28) have identified a Syrian pMCT-1 TTGTGCTCTGCCTTCCCCGTGACGTCA hamster cDNA clone (pl3-2A8) that is expressed at a high P2A8 TTGTGCT*T*CC*T*CCCGT**CG*CA level in early G1 but rapidly decreases by late G1. The sequence is highly repetitive in the Syrian hamster genome TAGATG TA and gives rise to a heterogeneous population of transcripts. FIG. 5. Sequence comparison of pMCT-1 and a Gl-specific Because of these similarities with the mouse IAP elements, Syrian hamster cDNA clone. The 160-nucleotide sequence of pl3- we compared the sequence of the 605 nucleotides of our 2A8, a cloned cDNA specifically expressed in G1 in Syrian hamster cloned IAP LTR cDNA, pMCT-1 (ref. 20; unpublished cells (28), was compared in both orientations to the 605-nucleotide sequence of the TAP LTR cDNA pMCT-1. Two regions of partial data), with the 160 nucleotides of the Syrian hamster G1- homology were found. Dashes indicate the absence of nucleotides in specific cDNA clone, P2A8 (28). Two regions of limited p13-2A8; asterisks indicate sequence mismatches, and carets indi- homology were found and are shown in Fig. 5 along with the cate nucleotides that must be deleted from p13-2A8 to keep the differences between the two sequences in these regions. sequence in register with pMCT-1. These deleted nucleotides are Considering the species difference and considering that both shown in the line beneath the caret. Biochemistry: Augenlicht and Halsey Proc. Natl. Acad. Sci. USA 82 (1985) 1949 The alterations in the level of IAP LTR transcripts may A. M., Bishop, J. M. & Varmus, H. E. (1981) Cell 23, involve changes in RNA stabilization as well as gene tran- 311-322. scription, since the level of hybridization falls so rapidly 6. Fung, Y.-K. T., Lewis, W. G., Crittenden, L. B. & Kung, (Fig. 2 Lower) and the hybridization continues to increase H. J. (1983) Cell 33, 357-368. 7. Nusse, R., van Ooyen, A., Cox, D., Fung, Y.-K. T. & while the cells remain arrested from day 4 through day 6 Varmus, H. (1984) Nature (London) 307, 131-136. (Fig. 2 Upper). It might be expected that sequences that are 8. Nusse, R. & Varmus, H. E. (1982) Cell 31, 99-109. temporally regulated in the cell cycle would require a rela- 9. Dickson, C., Smith, R., Brookes, S. & Peters, G. (1984) Cell tively short half-life in cycling cells, and Hirschhorn et al. 37, 529-536. have found that the Gl-specific sequence P13-2A8 turns over 10. Bernhard, W. (1960) Cancer Res. 20, 712-727. rapidly (28). 11. Lueders, K. K., Segal, S. & Kuff, E. L. (1977) Cell 11, 83-94. The experiments reported here do not permit us to con- 12. Lueders, K. K. & Kuff, E. L. (1977) Cell 12, 963-972. clude that the sequences responsible for cell cycle-specific 13. Biczysko, W., Pienkowski, M., Solter, D. & Koprowski, H. regulation reside within the LTR. Since the LTRs contain (1973) J. Natl. Cancer Inst. 51, 1041-1050. 14. Calarco, P. G. & Szollosi, D. (1973) Nature (London) New transcriptional regulatory sequences, we believe that this is Biol. 243, 91-93. likely. It should be noted, for example, that sequences 15. Chase, D. G. & Piko, L. (1973) J. Natl. Cancer Inst. 51, responsible for glucocorticoid regulation of MMTV expres- 1971-1975. sion are located within the MMTV LTR (37). It is also 16. Piko, L., Hammons, M. D. & Taylor, K. D. (1984) Proc. Natl. possible that related cell cycle-specific sequences exist in the Acad. Sci. USA 81, 488-492. genome unassociated with viral-related genes. 17. Kuff, E. L., Wivel, N. A. & Lueders, K. K. (1968) Cancer It has been suggested that IAP LTRs are moveable genetic Res. 28, 2137-2148. elements capable of affecting transcription of other genes 18. Sato, T., Friend, C. & deHarven, E. (1971) Cancer Res. 31, with which they are associated or at sites to which they are 1402-1417. 19. Kuff, E. L., Lueders, K. K., Ozer, H. L. & Wivel, N. A. transposed (38). It is interesting to consider whether the (1972) Proc. Natl. Acad. Sci. USA 69, 218-222. linkage of genes to these LTRs also would place their 20. Augenlicht, L. H., Kobrin, D., Pavlovec, A. & Royston, expression under cell-cycle regulation, due to either tran- M. E. (1984) J. Biol. Chem. 259, 1842-1847. scriptional promoter and enhancer sequences in the LTR 21. Augenlicht, L. H. & Kobrin, D. (1982) Cancer Res. 42, (refs. 20 and 39; unpublished data) or subsequent stabiliza- 1088-1093. tion of the transcript. This is of particular interest in the case 22. Royston, M. E. & Augenlicht, L. H. (1983) Science 222, of two myelomas, where IAP LTRs have been transposed 1339-1341. into the sequence c-mos, resulting in c-mos expression 23. Rechavi, G., Givol, D. & Canaani, E. (1982) Nature (London) (23-26). Since c-mos has been reported recently to have 300, 607-611. 24. Kuff, E. L., Feenstra, A., Lueders, K., Rechavi, G., Givol, D. to a yeast gene whose expression is & Canaani, E. (1983) Nature (London) 302, 547-548. necessary for yeast cells to be released from a G1 block (40), 25. Cohen, J. B., Unger, T., Rechavi, G., Canaani, E. & Givol, D. placing such a mouse gene under the influence of a regula- (1983) Nature (London) 306, 797-799. tory element that is active in G1 could determine whether the 26. Gattoni-Celli, S., Hsiao, W.-L. W. & Weinstein, I. B. (1983) cells continuously divide or arrest in G1. It is also of interest Nature (London) 306, 795-797. to consider that activation of other proto-onc genes by other 27. Kirschmeier, P., Gattoni-Celli, S., Dina, D. & Weinstein, I. B. viral LTRs, such as c-myc, c-erbB and the intl and int2 loci (1982) Proc. Natl. Acad. Sci. USA 79, 2773-2777. (3-9), could be linked to early G1 because of a cell-cycle 28. Hirschhorn, R. R., Aller, P., Yuan, Z.-A., Gibson, C. W. & specificity of the LTR. Baserga, R. (1984) Proc. Natl. Acad. Sci. USA 81, 6004-6008. 29. Gambari, R., Marks, P. A. & Rifkind, R. A. (1979) Proc. Natl. In a broader sense, the question may be raised as to Acad. Sci. USA 76, 4511-4515. whether repetitive sequence elements, such as those se- 30. Darzynkiewicz, Z., Williamson, B., Carswell, E. A. & Old, quences in the IAP LTR that share homology with the L. J. (1984) Cancer Res. 44, 83-90. Gl-specific Syrian hamster repetitive sequence, are related 31. White, B. A. & Bancroft, F. C. (1982) J. Biol. Chem. 257, to coordinate cell cycle-specific expression of many genes, 8569-8572. especially in early development when the IAP gene family is 32. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) normally active. This would be similar to the hypothesis J. Mol. Biol. 113, 237-251. recently put forth by Rigby and colleagues that a repetitive 33. Rougeon, F. & Mach, B. (1977) Gene 1, 229-240. mouse genomic element is responsible for the coordinate 34. Friedman, E. & Schildkraut, C. L. (1978) Proc. Natl. Acad. expression of gene Sci. USA 75, 3813-3817. sets in early development and their 35. Harrison, P. R. (1977) Int. Rev. Biochem. 15, 227-267. reexpression in transformed mouse cells (41, 42). 36. Linzer, D. I. H. & Nathan, D. (1983) Proc. Natl. Acad. Sci. USA 80, 4271-4275. We thank Z. Darzynkiewicz for assistance with flow cytometry; 37. Buetti, E. & D. Kobrin, L. Ngo, P. Marks, and R. Rifiind for their help; and R. Diggelmann, H. (1983) EMBO J. 2, 1423-1429. 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