J. Biochem. 110, 146-150 (1991)
Down Modulation of N-myc, Heat-Shock Protein 70, and Nucleolin during the Differentiation of Human Neuroblastoma Cells1 Tadamasa Murakami,**'* Hisamitsu Ohmori,* Sadao Gotoh,* Tohru Tsuda,* Ryoichi Ohya,** Shinobu Akiya,*** and Ken Higashi* 2
Received for publication, January 7, 1991
Cultured human neuroblastoma (GOTO) cells were induced to differentiate by dibutyryl cyclic AMP (BtacAMP) and/or retinoic acid (RA). A combination of BtjcAMP (1 mM) and RA (1 /*M) yielded the most significant networks of neurites after 3 to 4 days, this being associated with the reduction of N-myc mRNA levels. Next, we examined several cellular genes that were possibly linked with changes in N-myc gene expression under these conditions. Among the genes examined, both nucleolin and a major heat-shock protein (hsp70) mRNA8 showed changes concomitant with those in N-myc mRNA levels when induced by BtjcAMP and RA. Dibutyryl cAMP alone induced several short cellular processes and caused a marked decrease in N-myc mRNA within 2 days. RA alone induced a few long and straight neurites along the longitudinal axis of individual cells and a significant decrease in growth rate but showed neither network formation nor a decrease in N-myc gene expression. These results indicate differential effects of BtjcAMP and RA on the regulatory mechanisms of both cell proliferation and differentiation and also indicate a possible association of expression of N-myc gene with those of hsp70 and nucleolin genes.
Down regulation of N-myc gene expression in several neuroblastoma cell lines has been induced by retinoic acid (RA) (2-3). However, not all neuroblastoma cell lines respond to RA in a similar manner. On the other hand, dibutyryl cyclic AMP (BtjcAMP) is widely used to induce the differentiation of several cell lines (4, 5), but its effect is unstable in some cases and frequently reversible after removal of BtjcAMP from the culture medium (6, 7). Recently, a combination of several inducers was used successfully to induce cell differentiation {8-10). In the present study, we used BtjcAMP and RA either alone or in combination to induce the differentiation of neuroblastoma GOTO cells and found that these inducers had different effects on cell proliferation and differentiation. Under these different conditions, we had an opportunity to investigate which cellular genes might be linked with the changes in N-myc gene expression. Previously, we reported that N-myc amplification in the neuroblastoma was closely related to the worst prognosis (11). Overexpression of the N-myc product may contribute to a malignant phenotype of neuroblastoma, such as rapid cell growth, high rate of metastasis, or avoidance of immunological suppression by the host (12). We examined the changes in the expression of several possible genes which might be related to these malignant phenotypes. Among the genes examined, both nucleolin (13) and a major heat-shock protein (hsp70) (14) 1 This study was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan. 1 To whom correspondence should be addressed. Abbreviations: BtjcAMP, dibutyryl cyclic AMP; RA, retinoic acid; PBS, phosphate-buffered saline; hsp70, heat-shock protein 70; MHC, major histocompatibility complex.
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were closely associated with the changes of N-myc expression under different cellular conditions induced by RA and BkcAMP either alone or in combination. These results suggest that nucleolin and hsp70 may play a role in the rapid proliferation of neuroblastoma cells. MATERIALS AND METHODS Chemicals—BtjcAMP (2V»,O2'-dibutyryl adenosine 3': 5'-monophosphate) and all- trans-BA were obtained from Sigma Chemical (St. Louis, MO). [>-"P]dCTP was purchased from New England Nuclear (111 TBq/mmol). All- trans-RA was dissolved in ethanol to a concentration of 5 mM and stored at -20*C. Cell Culture—GOTO cells (25) and NB-1 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo). GOTO cells were grown in RPMI 1640 supplemented with glutamine (2 mM), penicillin (50 units/ ml), streptomycin (50/ig/ml), and 10% fetal calf serum (Gibco Lab., NY) in a humidified atmosphere of 95% air and 5% CO,. Cells were plated at 1-2 x 105 cells/ml/cm 2 . After a 24 h subculture, cells were maintained in RPMI 1640 containing 10% FCS and either 1 mM BtjcAMP or 1 /*M RA or a combination of both reagents, or without either. For treatment longer than 3 days, a one-third volume of culture medium containing the same reagent(s) was added without exchanging the whole medium. Cells from five to six culture plates (diameter 140 mm, Falcon, CA) were used for one RNA extraction under given conditions at a given time. Northern Blots—Total cellular RNA was isolated from neuroblastoma cells by using the guanidinium thiocyanate procedure (16). Twenty micrograms of total RNA was electrophoresed on 1% agarose (SeaKem, ME) under J. Biochem.
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Departments of "Biochemistry, "Oral Surgery, and '"Ophthalmology, School of Medicine, University of Occupational and Environmental Health, Yahatanishi-ku, KUakyushu, Fukuoka 807
Regulation of N-myc, Hsp70, and Nucleolin Genes
RESULTS Induction of Differentiation of Neuroblastoma Cells— The induction of differentiation was monitored by the microscopic observation of neurite outgrowth and changes in N-myc mRNA levels. We used neuroblastoma GOTO
Fig. 1. Morphological changes In GOTO cells Induced by Bt,cAMP and RA in combination or each reagent alone. A) GOTO cells, control; B) Bt*cAMP and RA, 4 days; C) Bt^cAMP alone, 4 days; D) RA alone, 4 days. Vol. 110, No. 1, 1991
cells for the following reasons: this is a widely available cell line, it shows little aggregation during cultivation (this is important for observing neurite extension and also to evaluate the percentages of cell populations that differentiate) , and it has been thoroughly characterized (15). The best procedure so far found for inducing remarkable neurite outgrowth and network formation, as shown in Fig. 1, is the use of Bt^cAMP (1 mM) and RA (1 //M) in combination. The Northern blot analysis of N-myc mRNA during the differentiation of GOTO cells after treatment with Bt^.cAMP and RA is shown in Fig. 2. The reduction of N-myc mRNA content became distinct after 2 days, during which time morphological changes also became clearly detectable. After the establishment of this down-regulation system for N-myc gene, examinations were made to determine whether other cellular genes also changed their expression during the change in N-myc gene expression. We examined the changes in the expression of the following genes; c-fos, c-myc, cathepsin L (23), class I MHC antigen (24), fibronectin, lnminin, nucleolin, and several heat-shock proteins. Both nucleolin and hsp70 gene expressions were altered significantly. Changes in Nucleolin and Hsp70 Gene Expression—As shown in Fig. 3, nucleolin mRNA decreased gradually with time during the induction of GOTO cell differentiation by BtjcAMP and RA. The behavior of hsp70 mRNA was rather complex. Hsp70 mRNA decreased once in the early period of treatment and then slightly increased and then again declined. This pattern of hsp70 mRNA was reproducible during the induction of GOTO cell differentiation by both reagents and was also observed in other neuroblastoma NB-1 cells (data not shown). Thus, Bt2cAMP and RA in combination caused remarkable neurite outgrowth and reduction of N-myc mRNA, coupled with suppression of nucleolin and hsp70 mRNA levels. From these results, however, we cannot determine whether the reduction of nucleolin and hsp70 was coincidental, being due to the induction of differentiation but not
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denaturing conditions (17) and transferred to nylon filters (Hybond N, Amersham, U.K.). The filters were hybridized with 32P-labeled nick-translated probes. Hsp70 [human, BamHl-EcoBl fragment, 0.8 kb, provided by Dr. Joseph R. Nevins (18)], nucleolin [mouse, Pstl fragment, 0.42 kb, provided by Dr. Francois Amalric (19)], N-myc [human, EcoRI-BamHl fragment, 1.0 kb, provided by Dr. Nancy E. Kohl (20)], and £-actin [chick, Pstl fragment, 1.8 kb (21)], were used as probes. Hybridization was carried out in 4X SSC, 50% formamide at 42'C for either 18 or 45 h. The filters were washed under mild conditions (2xSSC, 0.1% SDS for 30 min and 1 x SSC, 0.1% SDS for 30 min, twice at 42*C) for nucleolin or highly stringent conditions (0.2 x SSC, 0.1% SDS at 60"C for 30 min three times) for hsp70, N-myc, and /?-actin. Quantitation of mRNA Expression—The content of individual mRNA was estimated by densitometric scanning of autoradiographic film and normalized with respect to the signals obtained with a 32P-labeled /3-actin probe. Analysis of Cell Cycle Profiles—GOTO cells were treated with individual inducers for 3 days and cell-cycle profiles were evaluated by a cell sorter (EPICS 752, Coulter, FL). Cells were washed twice with PBS and twice with 1 ml of 0.1% sodium citrate at 4°C. They were again suspended in 1 ml of 0.1% sodium citrate containing 0.2% Nonidet P-40 (Shell Chemical, NY) and 0.25 mg of RNase A (Sigma Chemical, MO) and incubated for 30 min at 4'C. The crude nuclear fraction was collected by centrifugation and suspended in 0.5 ml of 0.1% sodium citrate containing propidium iodide (50/*g/ml). After filtration of the crude nuclei through a nylon mesh (40//m), cell-cycle profiles were determined by the use of a cell sorter (22).
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T. Murakami et aL
i 20 • 2 8 )•
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o h -/3-actin
Fig. 2. Northern blot analysis of the expression of the N-myc gene during induction of differentiation by Bt,cAMP and RA. Total RNA was prepared on a given day and 20 m was processed for Northern blot analysis as described in "MATERIALS AND METHODS."
O — O N-myc A • A Hsp 70 X - - X Nudeolin
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/ 100
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\
\
\
v A.
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E
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Control RA BtjcAMP Bt2cAMP + RA
1 2 3 4 days Culture Time After Treatment
Fig. 4. Time coarse of cell numbers daring treatment with individual inducers. Cell numbers were directly determined under a light microscope in triplicate dishes by two persons. The ordinate is a logarithmic scale. Standard deviations are shown by the vertical bars for the results at 4 days.
\
\
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4 days
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Fig. 3. Contents of N-myc, nucleolin, and hsp70 mRNA during Induction of differentiation by Bt,cAMP and RA. One day after seeding of GOTO cells, differentiation was induced by the addition of both Bt,cAMP (lmM) and RA (l^M). Values are expressed as percentages of control cells after normalization with respect to /3-actin mRNA. (C) Control value, no induction.
a consequence of N-myc reduction, or merely due to the decreased growth rate of GOTO cells. A number of regulatory mechanisms must be involved in the gene expression of both nucleolin and hsp70. Interestingly, we observed distinct characteristics of different inducers in this study. Effects of Different Inducers on GOTO CeUs—Dibutyryl cAMP alone induced multiple neurite-like processes of GOTO cells 2 to 4 days after the start of treatment, although network formation was rarely detected under these conditions (Fig. 1C). Suppression of the growth rate by Bt^cAMP alone was not significant, particularly during the early period of differentiation (Fig. 4). The incorporation of [ 3 H]thymidine and/or [32P]orthophosphate into cellular DNA showed no suppression of DNA replication by BtjcAMP alone within 2 days after treatment (Murakami, unpublished). RA alone suppressed the proUfication of GOTO cells most significantly (Figs. 4 and 5) and induced
only one or two long and straight neurites along the longitudinal axis of individual cells (Fig. ID). Both reagents together reduced the growth rate moderately, but induced the most significant networks of neurites associated with many growth cones (Figs. 1 and 4). Average cell numbers as percentages of the control in individual dishes at 4 days after individual inductions were as follows: 81% for B^cAMP alone, 61% for BtjcAMP plus RA, and 54% for RA alone. Profiles of DNA content by cytofluorometry at 3 days after the start of treatment by individual inducers are shown in Fig. 5. The most significant accumulation of Gl phase was detectable after treatment with RA alone. Accumulations of G2 + M phase after treatment with either BtjcAMP plus RA or BtjcAMP alone were significant at 3 days and were partially due to the inhibition of mitosis by elevated levels of intracellular cyclic AMP (25, 26). The results of cell number counting (shown in Fig. 4) were essentially comparable with those obtained by cytofluorometry. Next, we investigated relationships among morphological differences, growth rates, and the expressions of N-myc and other genes. Surprisingly, BtjcAMP alone did not suppress significantly the growth rate of GOTO cells under these conditions, but reduced most remarkably N-myc gene expression, that is, to levels of approximately one-third of the control at 3 days (Figs. 6 and 7). RA alone decreased significantly the growth rate of GOTO cells, but had no effect on the reduction of N-myc gene expression. These results indicate that the'reduction of N-myc mRNA is not merely due to the decrease in growth rate. No significant changes in c-myc expression were observed under these conditions for differentiation, although the J. Biochem.
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-N-myc
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Regulation of N-myc, Hsp70, and Nucleolin Genes
G1
S + G2 + M BfecAMP
s RA
+ RA
DNA Content
Fig. 5. DNA histogram of GOTO cells during treatment of Individual inducere. Cell-cycle profiles of GOTO cells were analyzed by a cell sorter (EPICS 752) as described in "MATERIALS AND METHODS." Percentages of S + G2+M phase are 67% for control, 78% for Bt^AMP alone, 47% for RA alone, and 55% for Bt,cAMP plus RA. Duplicate samples for each case showed, essentially the same pattern. Ordinate, arbitrary unit of cell population; abscissa, DNA content.
C
Bt^AlP Bt,cAIP RA
+
RA
O — O N-myc A- AHsp 70 X - - X Nudeolin
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expression of c-myc gene was rather low compared to that of N-myc gene in GOTO cells (data not shown). Coordinate Expression of N-myc, Nucleolin, and Hsp70 Genes—Interestingly, the coordinate gene expression of N-myc, nucleolin, and hsp70 was detected under these conditions. As shown in the time-course study of Bt^cAMP alone (Fig. 7), N-myc, nucleolin, and hsp70 mRNAs all decreased simultaneously after treatment with BtjcAMP alone. N-myc mRNA started to decrease even at one day after treatment and gradually declined thereafter, in contrast to the results obtained with BkcAMP plus RA (Fig. 3). Although no decrease of N-myc, hsp70, or nucleolin mRNA was caused by treatment with RA alone, the level of hsp70 mRNA was rather increased on the third day (Fig. 6). The different profiles of the time course of these mRNAs between Figs. 3 and 7 are thus due to either the presence or absence of RA. To sum up the results, the overall profiles of changes in N-myc, hsp70, and nucleolin mRNA after individual treatment were essentially the same (Fig. 6); that is, they behaved coordinately. DISCUSSION In the present study, we focused attention on i) differential effects of BtjcAMP and RA on the expression of N-myc, neurite outgrowth, and growth arrest in neuroblastoma cells and ii) coordinate expression of N-myc and other Vol. 110, No. 1, 1991
50
0 (c)
days Days After Treatment
Fig. 7. Time course of the levels of N-myc, hsp70, and nucleolin mRNA after treatment of GOTO cells with Bt,cAMP alone. Experimental procedures were essentially the same as those in Fig. 2.
cellular genes. A number of studies have been reported on the induction of differentiation of neuroblastoma cells by various reagents {1-10). The extension of neurite -like cellular processes could be observed after treatment of neuroblastoma cells with various inducere and used as a marker of differentiation. Down regulation of N-myc gene expression in several neuroblastoma cell lines has been induced by RA (1-3) and is also considered as a marker of differentiation of neuroblastoma cells. Cell growth and differentiation are usually mutually
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Fig. 6. Changes in the expression of N-myc, hsp70, and nucleolin genes on treatment of GOTO cells with individual inducere. GOTO cells were treated either with a combination of BtjcAMP+RA, or BticAMP alone or RA alone for 3 days and their RNAs were analy*ed by Northern blot analysis. Values are expressed as percentages of those of control cells. Four to seven experiments were carried out and standard deviations are shown by the thin lines on the individual bars.
Control
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products have possible roles in supporting cell growth, at least in part via the stimulation of protein biosynthesis as a consequence of an increase of ribosomes. Of course, bsp70 is a multifunctional protein and may affect other cellular functions besides the activation of nucleolar activity. We are grateful to Drs. J.R. Nevins, F. Amalric, and N.E. Kohl for kindly providing the probes, Drs. H. Hirano and T. Sugiura for technical advice. Misses A. Furutani and K. Nishiraura for technical assistance, Dr. T. Iwasaki for statistical evaluation and the Japanese Cancer Research Resources Bank for providing the neuroblastoma cell lines. REFERENCES 1. Thiele, C.J., Reynolds, C.P., & Israel, M.A. (1985) Nature 313, 404-406 2. Amatsuda, T.T., SideU, N., Ranyard, J., & Koeffler, H.P. (1985) Biochem. Biophys. Res. Commun. 126, 1189-1195 3. Horii, Y., Sugimoto, T., Imanishi, J., Tsuboi, K., & Hatanaka, M. (1989) Int. J. Cancer 43, 305-309 4. Prasad, K.M., Sahu, S.K., & Sinha, P.K. (1976) J. Nad. Cancer InsL 57, 619-629 5. Rupniak, H.T., Rein, G., Powell, J.F., Ryder, T.A., Carson, S., Povey, S., & Hill, B.T. (1984) Cancer Res. 44, 2600-2607 6. Morita, A., Kitagawa, Y., & Sugimoto, E. (1985) J. Cell. Physiol. 125,263-272 7. Macintyre, E.H., Wintersgill, C.J., Perkins, J.P., & Vatter, A.E. (1972) J. Cell Sci. 11, 639-667 8. SideU, N. & Koeffler, H.P. (1988) Cancer Res. 48, 2226-2230 9. Lando, M., Abemayor, E., Verity, M.A., & SideU, N. (1990) Cancer Res. 50, 722-727 10. Rocci, P., Simone, G., Lorenzetti, A., Granchi, D., Paolucci, P., & Paolucci G. (1980) Anticancer Res. 9 1605-1610 11. Tsuda, T., Obara, M., Hirano, H., Gotoh, S., Kubomura, N., Higashi, K., Kuroiwa, A., Nakagawara, A., Nagahara, N., & Shlmizu, K. (1987) Cancer 60, 820-826 12. NoweU, P.C. (1986) Cancer Res. 46, 2203-2207 13. Jordan, G. (1987) Nature 329, 489-490 14. Pelham, H.R.B. (1986) Cell 46, 959-961 15. Sekiguchi, M., Oota, T., Sakakibara, K., Inui, N., & Fujii, G. (1979) Jpn. J. Exp. Med. 49, 67-83 16. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., & Rutter, W.J. (1979) Biochemistry 18, 5294-5299 17. Lehrach, H., Diamond, D., Wozney, J.M., &Boedtker, H. (1977) Biochemistry 16, 4743-4751 18. Kao, H.T. & Nevins, J.R. (1983) Mol. Cell. Biol. 3, 2058-2065 19. Bourbon, H.-M.,Prudhomme, M.,& Amalric, F. (1988) GeneOS, 73-84 20. Kohl,N.E.,Legouy,E.,Depinho,RA.,Nisen,P.D.,Smith,R.K., Gee, C.E., & Alt, F.W. (1986) Nature 319, 73-77 21. Kost, D.R., Theodrakis, N., & Hughes, S.H. (1983) Nucleic Acids Res. 11, 8287-8301 22. Parks, D.R. & Herzenberg, L.A. (1984) in Methods in Enzymology (Di Sabato, G., Langone, J.J., & Van Vunakis, H.,eds.) Vol. 108, pp. 197-241, Academic Press, New York 23. Troen, B.R., Gal, S., &Gottesman, M.M. (1987) Biochem. J. 246, 731-735 24. Bernards, R, Dessain, S.K., & Weinberg, RA. (1986) Cell 47, 667-674 25. Dumont, J.E., Jauniaux, J.C., &Roger, P.P. (1989) TIBS 14,6771 26. Boynton, A.L. & Whitfield, J.F. (1983) Anna. Rev. Biochem. 52, 193-294 27. La Rocca, S.A., Grossi, M., Falcone, G., Alema, S., & Tat6, F. (1989) Cell 58, 123-131 28. Blau, H.M. (1988) Cell 53, 673-674 29. Akeson, R & Bernards, R (1990) Mol Cell. Biol 10, 2012-2016 30. Bates, S.E., Mickley, L.A., Chen, Y.-H., Richert, N., Rudick, J., Biedler, J.J., & Fojo, A.T. (1989) Mol Cell Biol. 9, 4337-4344 31. Thiele, C.J., Cohen, P.S., & Israel, M.A. (l988) Mol. Cell Biol. 8, 1677-1683 32. Lapeyre, B., Bourbon, H., & Amalric, F. (1987) Proa Natl. Acad. So. U.S.A. 84, 1472-1476 33. Ohmori, H., Murakami, T., Furutani, A., Higashi, K., Hirano, H., Gotoh, S., Masui, A., Nakamura, T., & Amalric, F. (1990) Exp. Cell Res. 189, 227-232 34. Pelhain, H.R.B. (1984) EMBO J. 3, 3095-3100 35. Kingston, RE., Baldwin, A.S., Jr., & Sharp, P.A. (1984) Nature 312, 280-282 36. Wu, B.J. & Morimoto, R.I. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6070-6074
J. Biochem.
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exclusive. It has been reported that transformation of myoblasts by retrovirus containing the myc oncogene inhibited differentiation, thus preventing cells from withdrawing from the cell cycle (27). MyoDl regulates myogenesis {28). The MyoDl protein, a nuclear phosphoprotein somewhat similar to the myc family of proteins, is a DNA-binding protein. In the present study, RA suppressed the growth of GOTO cells significantly, but little reduction of N-myc expression was detected after RA-treatment. On the other hand, Bt^cAMP significantly suppressed the expression of the N-myc gene without any decrease of growth rate of GOTO cells during the early stage of the induction of differentiation. Since GOTO cells responded to RA treatment, they have RA receptors. However, RA alone was not sufiicient to induce the most remarkable formation of neurite networks in GOTO cells. Bt^cAMP alone was also insufficient to induce stable neurite and network-formation, although it reduced the level of N-myc mRNA significantly. Synergistic action of RA and Bt^cAMP could induce significant morphological differentiation during a rather short period of treatment (Fig. 1). These results indicated that inhibition of DNA synthesis seems to be controlled separately from morphological differentiation. In other words, one or more mechanism(s) control induction of differentiation and others initiate or maintain the growth-arrested stage of the differentiating cells. The expression of N-myc in the neuroblastoma cells has been reported to down-regulate class I MHC antigen (24), neural cell adhesion molecule (NCAM) (29), and multidrug resistance gene (30). Moreover, the expressions of ppfiO0""10, p53 and c-myb genes are modulated by the N-myc gene product (31). Down regulation of N-myc gene expression might be associated with the increase of these gene expressions. For rapid cell growth, two groups of genes could be considered. One group is related to DNA replication and the other is related to protein biosynthesis. In our study, we detected coordinate changes of nucleolin and hsp70 mRNAs with N-myc mRNA levels. Nucleolin is a major nucleolar protein and has been found to be involved in the activation of ribosomal gene expression, ribosomal RNA processing, and ribosome assembly (23, 32). Nucleolin is a multifunctional protein and contributes to the control of ribosome biosynthesis at various steps (23). The coordinate expression of N-myc and nucleolin genes, therefore, seems to support rapid cell growth. Previously, we reported the simultaneous activation of nucleolin and hsp70 genes during the prereplicative stage of regenerating rat liver and primary cultured hepatocytes, in which the nucleolar chromatin must be reorganized for the stimulation of rDNA transcription (33). Hsp70 has a characteristic nucleolar location after heat shock. The expression of high levels of hsp70 also results in a more rapid recovery of normal nucleolar morphology after heat shock (14, 34). Kingston et al (35) suggested the regulation of hsp70 gene expression by c-myc gene product, the function of which might be similar to that of N-myc protein. Another report indicated possible roles of hsp7O protein in the regulation of cell growth (36). There are several subtypes of hsp70 in mammalian cells. Some are heatinducible, but others are expressed constitutively in growing cells (14). It is unknown, however, which type of hsp70 changed during the induction of differentiation reported in this paper. It seems likely that both nucleolin and hsp70
T. Murakami et al.
Down Modulation of N-myc, Heat-Shock Protein 70, and Nucleolin during the Differentiation of Human Neuroblastoma Cells1 Tadamasa Murakami,**'* Hisamitsu Ohmori,* Sadao Gotoh,* Tohru Tsuda,* Ryoichi Ohya,** Shinobu Akiya,*** and Ken Higashi* 2
Received for publication, January 7, 1991
Cultured human neuroblastoma (GOTO) cells were induced to differentiate by dibutyryl cyclic AMP (BtacAMP) and/or retinoic acid (RA). A combination of BtjcAMP (1 mM) and RA (1 /*M) yielded the most significant networks of neurites after 3 to 4 days, this being associated with the reduction of N-myc mRNA levels. Next, we examined several cellular genes that were possibly linked with changes in N-myc gene expression under these conditions. Among the genes examined, both nucleolin and a major heat-shock protein (hsp70) mRNA8 showed changes concomitant with those in N-myc mRNA levels when induced by BtjcAMP and RA. Dibutyryl cAMP alone induced several short cellular processes and caused a marked decrease in N-myc mRNA within 2 days. RA alone induced a few long and straight neurites along the longitudinal axis of individual cells and a significant decrease in growth rate but showed neither network formation nor a decrease in N-myc gene expression. These results indicate differential effects of BtjcAMP and RA on the regulatory mechanisms of both cell proliferation and differentiation and also indicate a possible association of expression of N-myc gene with those of hsp70 and nucleolin genes.
Down regulation of N-myc gene expression in several neuroblastoma cell lines has been induced by retinoic acid (RA) (2-3). However, not all neuroblastoma cell lines respond to RA in a similar manner. On the other hand, dibutyryl cyclic AMP (BtjcAMP) is widely used to induce the differentiation of several cell lines (4, 5), but its effect is unstable in some cases and frequently reversible after removal of BtjcAMP from the culture medium (6, 7). Recently, a combination of several inducers was used successfully to induce cell differentiation {8-10). In the present study, we used BtjcAMP and RA either alone or in combination to induce the differentiation of neuroblastoma GOTO cells and found that these inducers had different effects on cell proliferation and differentiation. Under these different conditions, we had an opportunity to investigate which cellular genes might be linked with the changes in N-myc gene expression. Previously, we reported that N-myc amplification in the neuroblastoma was closely related to the worst prognosis (11). Overexpression of the N-myc product may contribute to a malignant phenotype of neuroblastoma, such as rapid cell growth, high rate of metastasis, or avoidance of immunological suppression by the host (12). We examined the changes in the expression of several possible genes which might be related to these malignant phenotypes. Among the genes examined, both nucleolin (13) and a major heat-shock protein (hsp70) (14) 1 This study was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan. 1 To whom correspondence should be addressed. Abbreviations: BtjcAMP, dibutyryl cyclic AMP; RA, retinoic acid; PBS, phosphate-buffered saline; hsp70, heat-shock protein 70; MHC, major histocompatibility complex.
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were closely associated with the changes of N-myc expression under different cellular conditions induced by RA and BkcAMP either alone or in combination. These results suggest that nucleolin and hsp70 may play a role in the rapid proliferation of neuroblastoma cells. MATERIALS AND METHODS Chemicals—BtjcAMP (2V»,O2'-dibutyryl adenosine 3': 5'-monophosphate) and all- trans-BA were obtained from Sigma Chemical (St. Louis, MO). [>-"P]dCTP was purchased from New England Nuclear (111 TBq/mmol). All- trans-RA was dissolved in ethanol to a concentration of 5 mM and stored at -20*C. Cell Culture—GOTO cells (25) and NB-1 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo). GOTO cells were grown in RPMI 1640 supplemented with glutamine (2 mM), penicillin (50 units/ ml), streptomycin (50/ig/ml), and 10% fetal calf serum (Gibco Lab., NY) in a humidified atmosphere of 95% air and 5% CO,. Cells were plated at 1-2 x 105 cells/ml/cm 2 . After a 24 h subculture, cells were maintained in RPMI 1640 containing 10% FCS and either 1 mM BtjcAMP or 1 /*M RA or a combination of both reagents, or without either. For treatment longer than 3 days, a one-third volume of culture medium containing the same reagent(s) was added without exchanging the whole medium. Cells from five to six culture plates (diameter 140 mm, Falcon, CA) were used for one RNA extraction under given conditions at a given time. Northern Blots—Total cellular RNA was isolated from neuroblastoma cells by using the guanidinium thiocyanate procedure (16). Twenty micrograms of total RNA was electrophoresed on 1% agarose (SeaKem, ME) under J. Biochem.
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Departments of "Biochemistry, "Oral Surgery, and '"Ophthalmology, School of Medicine, University of Occupational and Environmental Health, Yahatanishi-ku, KUakyushu, Fukuoka 807
Regulation of N-myc, Hsp70, and Nucleolin Genes
RESULTS Induction of Differentiation of Neuroblastoma Cells— The induction of differentiation was monitored by the microscopic observation of neurite outgrowth and changes in N-myc mRNA levels. We used neuroblastoma GOTO
Fig. 1. Morphological changes In GOTO cells Induced by Bt,cAMP and RA in combination or each reagent alone. A) GOTO cells, control; B) Bt*cAMP and RA, 4 days; C) Bt^cAMP alone, 4 days; D) RA alone, 4 days. Vol. 110, No. 1, 1991
cells for the following reasons: this is a widely available cell line, it shows little aggregation during cultivation (this is important for observing neurite extension and also to evaluate the percentages of cell populations that differentiate) , and it has been thoroughly characterized (15). The best procedure so far found for inducing remarkable neurite outgrowth and network formation, as shown in Fig. 1, is the use of Bt^cAMP (1 mM) and RA (1 //M) in combination. The Northern blot analysis of N-myc mRNA during the differentiation of GOTO cells after treatment with Bt^.cAMP and RA is shown in Fig. 2. The reduction of N-myc mRNA content became distinct after 2 days, during which time morphological changes also became clearly detectable. After the establishment of this down-regulation system for N-myc gene, examinations were made to determine whether other cellular genes also changed their expression during the change in N-myc gene expression. We examined the changes in the expression of the following genes; c-fos, c-myc, cathepsin L (23), class I MHC antigen (24), fibronectin, lnminin, nucleolin, and several heat-shock proteins. Both nucleolin and hsp70 gene expressions were altered significantly. Changes in Nucleolin and Hsp70 Gene Expression—As shown in Fig. 3, nucleolin mRNA decreased gradually with time during the induction of GOTO cell differentiation by BtjcAMP and RA. The behavior of hsp70 mRNA was rather complex. Hsp70 mRNA decreased once in the early period of treatment and then slightly increased and then again declined. This pattern of hsp70 mRNA was reproducible during the induction of GOTO cell differentiation by both reagents and was also observed in other neuroblastoma NB-1 cells (data not shown). Thus, Bt2cAMP and RA in combination caused remarkable neurite outgrowth and reduction of N-myc mRNA, coupled with suppression of nucleolin and hsp70 mRNA levels. From these results, however, we cannot determine whether the reduction of nucleolin and hsp70 was coincidental, being due to the induction of differentiation but not
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denaturing conditions (17) and transferred to nylon filters (Hybond N, Amersham, U.K.). The filters were hybridized with 32P-labeled nick-translated probes. Hsp70 [human, BamHl-EcoBl fragment, 0.8 kb, provided by Dr. Joseph R. Nevins (18)], nucleolin [mouse, Pstl fragment, 0.42 kb, provided by Dr. Francois Amalric (19)], N-myc [human, EcoRI-BamHl fragment, 1.0 kb, provided by Dr. Nancy E. Kohl (20)], and £-actin [chick, Pstl fragment, 1.8 kb (21)], were used as probes. Hybridization was carried out in 4X SSC, 50% formamide at 42'C for either 18 or 45 h. The filters were washed under mild conditions (2xSSC, 0.1% SDS for 30 min and 1 x SSC, 0.1% SDS for 30 min, twice at 42*C) for nucleolin or highly stringent conditions (0.2 x SSC, 0.1% SDS at 60"C for 30 min three times) for hsp70, N-myc, and /?-actin. Quantitation of mRNA Expression—The content of individual mRNA was estimated by densitometric scanning of autoradiographic film and normalized with respect to the signals obtained with a 32P-labeled /3-actin probe. Analysis of Cell Cycle Profiles—GOTO cells were treated with individual inducers for 3 days and cell-cycle profiles were evaluated by a cell sorter (EPICS 752, Coulter, FL). Cells were washed twice with PBS and twice with 1 ml of 0.1% sodium citrate at 4°C. They were again suspended in 1 ml of 0.1% sodium citrate containing 0.2% Nonidet P-40 (Shell Chemical, NY) and 0.25 mg of RNase A (Sigma Chemical, MO) and incubated for 30 min at 4'C. The crude nuclear fraction was collected by centrifugation and suspended in 0.5 ml of 0.1% sodium citrate containing propidium iodide (50/*g/ml). After filtration of the crude nuclei through a nylon mesh (40//m), cell-cycle profiles were determined by the use of a cell sorter (22).
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Fig. 2. Northern blot analysis of the expression of the N-myc gene during induction of differentiation by Bt,cAMP and RA. Total RNA was prepared on a given day and 20 m was processed for Northern blot analysis as described in "MATERIALS AND METHODS."
O — O N-myc A • A Hsp 70 X - - X Nudeolin
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Fig. 4. Time coarse of cell numbers daring treatment with individual inducers. Cell numbers were directly determined under a light microscope in triplicate dishes by two persons. The ordinate is a logarithmic scale. Standard deviations are shown by the vertical bars for the results at 4 days.
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Fig. 3. Contents of N-myc, nucleolin, and hsp70 mRNA during Induction of differentiation by Bt,cAMP and RA. One day after seeding of GOTO cells, differentiation was induced by the addition of both Bt,cAMP (lmM) and RA (l^M). Values are expressed as percentages of control cells after normalization with respect to /3-actin mRNA. (C) Control value, no induction.
a consequence of N-myc reduction, or merely due to the decreased growth rate of GOTO cells. A number of regulatory mechanisms must be involved in the gene expression of both nucleolin and hsp70. Interestingly, we observed distinct characteristics of different inducers in this study. Effects of Different Inducers on GOTO CeUs—Dibutyryl cAMP alone induced multiple neurite-like processes of GOTO cells 2 to 4 days after the start of treatment, although network formation was rarely detected under these conditions (Fig. 1C). Suppression of the growth rate by Bt^cAMP alone was not significant, particularly during the early period of differentiation (Fig. 4). The incorporation of [ 3 H]thymidine and/or [32P]orthophosphate into cellular DNA showed no suppression of DNA replication by BtjcAMP alone within 2 days after treatment (Murakami, unpublished). RA alone suppressed the proUfication of GOTO cells most significantly (Figs. 4 and 5) and induced
only one or two long and straight neurites along the longitudinal axis of individual cells (Fig. ID). Both reagents together reduced the growth rate moderately, but induced the most significant networks of neurites associated with many growth cones (Figs. 1 and 4). Average cell numbers as percentages of the control in individual dishes at 4 days after individual inductions were as follows: 81% for B^cAMP alone, 61% for BtjcAMP plus RA, and 54% for RA alone. Profiles of DNA content by cytofluorometry at 3 days after the start of treatment by individual inducers are shown in Fig. 5. The most significant accumulation of Gl phase was detectable after treatment with RA alone. Accumulations of G2 + M phase after treatment with either BtjcAMP plus RA or BtjcAMP alone were significant at 3 days and were partially due to the inhibition of mitosis by elevated levels of intracellular cyclic AMP (25, 26). The results of cell number counting (shown in Fig. 4) were essentially comparable with those obtained by cytofluorometry. Next, we investigated relationships among morphological differences, growth rates, and the expressions of N-myc and other genes. Surprisingly, BtjcAMP alone did not suppress significantly the growth rate of GOTO cells under these conditions, but reduced most remarkably N-myc gene expression, that is, to levels of approximately one-third of the control at 3 days (Figs. 6 and 7). RA alone decreased significantly the growth rate of GOTO cells, but had no effect on the reduction of N-myc gene expression. These results indicate that the'reduction of N-myc mRNA is not merely due to the decrease in growth rate. No significant changes in c-myc expression were observed under these conditions for differentiation, although the J. Biochem.
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-N-myc
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Regulation of N-myc, Hsp70, and Nucleolin Genes
G1
S + G2 + M BfecAMP
s RA
+ RA
DNA Content
Fig. 5. DNA histogram of GOTO cells during treatment of Individual inducere. Cell-cycle profiles of GOTO cells were analyzed by a cell sorter (EPICS 752) as described in "MATERIALS AND METHODS." Percentages of S + G2+M phase are 67% for control, 78% for Bt^AMP alone, 47% for RA alone, and 55% for Bt,cAMP plus RA. Duplicate samples for each case showed, essentially the same pattern. Ordinate, arbitrary unit of cell population; abscissa, DNA content.
C
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expression of c-myc gene was rather low compared to that of N-myc gene in GOTO cells (data not shown). Coordinate Expression of N-myc, Nucleolin, and Hsp70 Genes—Interestingly, the coordinate gene expression of N-myc, nucleolin, and hsp70 was detected under these conditions. As shown in the time-course study of Bt^cAMP alone (Fig. 7), N-myc, nucleolin, and hsp70 mRNAs all decreased simultaneously after treatment with BtjcAMP alone. N-myc mRNA started to decrease even at one day after treatment and gradually declined thereafter, in contrast to the results obtained with BkcAMP plus RA (Fig. 3). Although no decrease of N-myc, hsp70, or nucleolin mRNA was caused by treatment with RA alone, the level of hsp70 mRNA was rather increased on the third day (Fig. 6). The different profiles of the time course of these mRNAs between Figs. 3 and 7 are thus due to either the presence or absence of RA. To sum up the results, the overall profiles of changes in N-myc, hsp70, and nucleolin mRNA after individual treatment were essentially the same (Fig. 6); that is, they behaved coordinately. DISCUSSION In the present study, we focused attention on i) differential effects of BtjcAMP and RA on the expression of N-myc, neurite outgrowth, and growth arrest in neuroblastoma cells and ii) coordinate expression of N-myc and other Vol. 110, No. 1, 1991
50
0 (c)
days Days After Treatment
Fig. 7. Time course of the levels of N-myc, hsp70, and nucleolin mRNA after treatment of GOTO cells with Bt,cAMP alone. Experimental procedures were essentially the same as those in Fig. 2.
cellular genes. A number of studies have been reported on the induction of differentiation of neuroblastoma cells by various reagents {1-10). The extension of neurite -like cellular processes could be observed after treatment of neuroblastoma cells with various inducere and used as a marker of differentiation. Down regulation of N-myc gene expression in several neuroblastoma cell lines has been induced by RA (1-3) and is also considered as a marker of differentiation of neuroblastoma cells. Cell growth and differentiation are usually mutually
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Fig. 6. Changes in the expression of N-myc, hsp70, and nucleolin genes on treatment of GOTO cells with individual inducere. GOTO cells were treated either with a combination of BtjcAMP+RA, or BticAMP alone or RA alone for 3 days and their RNAs were analy*ed by Northern blot analysis. Values are expressed as percentages of those of control cells. Four to seven experiments were carried out and standard deviations are shown by the thin lines on the individual bars.
Control
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products have possible roles in supporting cell growth, at least in part via the stimulation of protein biosynthesis as a consequence of an increase of ribosomes. Of course, bsp70 is a multifunctional protein and may affect other cellular functions besides the activation of nucleolar activity. We are grateful to Drs. J.R. Nevins, F. Amalric, and N.E. Kohl for kindly providing the probes, Drs. H. Hirano and T. Sugiura for technical advice. Misses A. Furutani and K. Nishiraura for technical assistance, Dr. T. Iwasaki for statistical evaluation and the Japanese Cancer Research Resources Bank for providing the neuroblastoma cell lines. REFERENCES 1. Thiele, C.J., Reynolds, C.P., & Israel, M.A. (1985) Nature 313, 404-406 2. Amatsuda, T.T., SideU, N., Ranyard, J., & Koeffler, H.P. (1985) Biochem. Biophys. Res. Commun. 126, 1189-1195 3. Horii, Y., Sugimoto, T., Imanishi, J., Tsuboi, K., & Hatanaka, M. (1989) Int. J. Cancer 43, 305-309 4. Prasad, K.M., Sahu, S.K., & Sinha, P.K. (1976) J. Nad. Cancer InsL 57, 619-629 5. Rupniak, H.T., Rein, G., Powell, J.F., Ryder, T.A., Carson, S., Povey, S., & Hill, B.T. (1984) Cancer Res. 44, 2600-2607 6. Morita, A., Kitagawa, Y., & Sugimoto, E. (1985) J. Cell. Physiol. 125,263-272 7. Macintyre, E.H., Wintersgill, C.J., Perkins, J.P., & Vatter, A.E. (1972) J. Cell Sci. 11, 639-667 8. SideU, N. & Koeffler, H.P. (1988) Cancer Res. 48, 2226-2230 9. Lando, M., Abemayor, E., Verity, M.A., & SideU, N. (1990) Cancer Res. 50, 722-727 10. Rocci, P., Simone, G., Lorenzetti, A., Granchi, D., Paolucci, P., & Paolucci G. (1980) Anticancer Res. 9 1605-1610 11. Tsuda, T., Obara, M., Hirano, H., Gotoh, S., Kubomura, N., Higashi, K., Kuroiwa, A., Nakagawara, A., Nagahara, N., & Shlmizu, K. (1987) Cancer 60, 820-826 12. NoweU, P.C. (1986) Cancer Res. 46, 2203-2207 13. Jordan, G. (1987) Nature 329, 489-490 14. Pelham, H.R.B. (1986) Cell 46, 959-961 15. Sekiguchi, M., Oota, T., Sakakibara, K., Inui, N., & Fujii, G. (1979) Jpn. J. Exp. Med. 49, 67-83 16. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J., & Rutter, W.J. (1979) Biochemistry 18, 5294-5299 17. Lehrach, H., Diamond, D., Wozney, J.M., &Boedtker, H. (1977) Biochemistry 16, 4743-4751 18. Kao, H.T. & Nevins, J.R. (1983) Mol. Cell. Biol. 3, 2058-2065 19. Bourbon, H.-M.,Prudhomme, M.,& Amalric, F. (1988) GeneOS, 73-84 20. Kohl,N.E.,Legouy,E.,Depinho,RA.,Nisen,P.D.,Smith,R.K., Gee, C.E., & Alt, F.W. (1986) Nature 319, 73-77 21. Kost, D.R., Theodrakis, N., & Hughes, S.H. (1983) Nucleic Acids Res. 11, 8287-8301 22. Parks, D.R. & Herzenberg, L.A. (1984) in Methods in Enzymology (Di Sabato, G., Langone, J.J., & Van Vunakis, H.,eds.) Vol. 108, pp. 197-241, Academic Press, New York 23. Troen, B.R., Gal, S., &Gottesman, M.M. (1987) Biochem. J. 246, 731-735 24. Bernards, R, Dessain, S.K., & Weinberg, RA. (1986) Cell 47, 667-674 25. Dumont, J.E., Jauniaux, J.C., &Roger, P.P. (1989) TIBS 14,6771 26. Boynton, A.L. & Whitfield, J.F. (1983) Anna. Rev. Biochem. 52, 193-294 27. La Rocca, S.A., Grossi, M., Falcone, G., Alema, S., & Tat6, F. (1989) Cell 58, 123-131 28. Blau, H.M. (1988) Cell 53, 673-674 29. Akeson, R & Bernards, R (1990) Mol Cell. Biol 10, 2012-2016 30. Bates, S.E., Mickley, L.A., Chen, Y.-H., Richert, N., Rudick, J., Biedler, J.J., & Fojo, A.T. (1989) Mol Cell Biol. 9, 4337-4344 31. Thiele, C.J., Cohen, P.S., & Israel, M.A. (l988) Mol. Cell Biol. 8, 1677-1683 32. Lapeyre, B., Bourbon, H., & Amalric, F. (1987) Proa Natl. Acad. So. U.S.A. 84, 1472-1476 33. Ohmori, H., Murakami, T., Furutani, A., Higashi, K., Hirano, H., Gotoh, S., Masui, A., Nakamura, T., & Amalric, F. (1990) Exp. Cell Res. 189, 227-232 34. Pelhain, H.R.B. (1984) EMBO J. 3, 3095-3100 35. Kingston, RE., Baldwin, A.S., Jr., & Sharp, P.A. (1984) Nature 312, 280-282 36. Wu, B.J. & Morimoto, R.I. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6070-6074
J. Biochem.
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exclusive. It has been reported that transformation of myoblasts by retrovirus containing the myc oncogene inhibited differentiation, thus preventing cells from withdrawing from the cell cycle (27). MyoDl regulates myogenesis {28). The MyoDl protein, a nuclear phosphoprotein somewhat similar to the myc family of proteins, is a DNA-binding protein. In the present study, RA suppressed the growth of GOTO cells significantly, but little reduction of N-myc expression was detected after RA-treatment. On the other hand, Bt^cAMP significantly suppressed the expression of the N-myc gene without any decrease of growth rate of GOTO cells during the early stage of the induction of differentiation. Since GOTO cells responded to RA treatment, they have RA receptors. However, RA alone was not sufiicient to induce the most remarkable formation of neurite networks in GOTO cells. Bt^cAMP alone was also insufficient to induce stable neurite and network-formation, although it reduced the level of N-myc mRNA significantly. Synergistic action of RA and Bt^cAMP could induce significant morphological differentiation during a rather short period of treatment (Fig. 1). These results indicated that inhibition of DNA synthesis seems to be controlled separately from morphological differentiation. In other words, one or more mechanism(s) control induction of differentiation and others initiate or maintain the growth-arrested stage of the differentiating cells. The expression of N-myc in the neuroblastoma cells has been reported to down-regulate class I MHC antigen (24), neural cell adhesion molecule (NCAM) (29), and multidrug resistance gene (30). Moreover, the expressions of ppfiO0""10, p53 and c-myb genes are modulated by the N-myc gene product (31). Down regulation of N-myc gene expression might be associated with the increase of these gene expressions. For rapid cell growth, two groups of genes could be considered. One group is related to DNA replication and the other is related to protein biosynthesis. In our study, we detected coordinate changes of nucleolin and hsp70 mRNAs with N-myc mRNA levels. Nucleolin is a major nucleolar protein and has been found to be involved in the activation of ribosomal gene expression, ribosomal RNA processing, and ribosome assembly (23, 32). Nucleolin is a multifunctional protein and contributes to the control of ribosome biosynthesis at various steps (23). The coordinate expression of N-myc and nucleolin genes, therefore, seems to support rapid cell growth. Previously, we reported the simultaneous activation of nucleolin and hsp70 genes during the prereplicative stage of regenerating rat liver and primary cultured hepatocytes, in which the nucleolar chromatin must be reorganized for the stimulation of rDNA transcription (33). Hsp70 has a characteristic nucleolar location after heat shock. The expression of high levels of hsp70 also results in a more rapid recovery of normal nucleolar morphology after heat shock (14, 34). Kingston et al (35) suggested the regulation of hsp70 gene expression by c-myc gene product, the function of which might be similar to that of N-myc protein. Another report indicated possible roles of hsp7O protein in the regulation of cell growth (36). There are several subtypes of hsp70 in mammalian cells. Some are heatinducible, but others are expressed constitutively in growing cells (14). It is unknown, however, which type of hsp70 changed during the induction of differentiation reported in this paper. It seems likely that both nucleolin and hsp70
T. Murakami et al.
