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Control of intracellular localization and degradation of c-FOS protein
228
C o l l o q u e "ContrSle M o l t c u l a i r e du C y c l e Cellulaire", Lyon, juillet 1992
MICIt, OSCOI'IC IMAGING OF T I l E CIIROMATIN IN LIVING CELLS - A P I L O T S T U D Y RONOT Xavier (1), COZ.ZIJean (2), GRUNrWALD Didier (3), ROBERT-NICOUD Michel (1), BRUGAL Gtrard (1), SELE Bernard (2). (I) Equipe de Reconnaissance des Formes et Microscopic Quantitative, Lab. "I1M3-1MAG, UniversitE J. Fourier, BP53X, F-38041 Grenoble Cedex. (2) Lab. de Biologie de la Reproduction, Facuh# de MEdecine. Domaine de la Merci , I:-38706 La Tronche Cedex. (3) DBMS, Lab. de Biologic Molt~culaire du Cycle Cellulaire, CENG, BP85X F-38041Grenoble Cedex.
CONTROL OF INTRACELLULAR LOCALIZATION D E G R A D A T I O N O F c-FOS P R O T E I N
AND
Seree C A R I L L O . Pierre ROUX, Ann-Muriel STEFF, Maryse E T I E N N E - J U L A N , J e a n - M a r i e B L A N C H A R D , Philippe JEANTEUR and Marc PIECHACZYK Laboratoire de Biologie Moldculaire. UA CNRS 1191 "GEndtique MolEculaire". place E. Baraillon -34095 - Montpellier Cede.r 05 France
One l~oal of cell biology is to explain interactions between molecules in a functtonal cell. The developments and applications of flow cytometry and fluorescence image cytometry have added new dimensions to knowledge about the structure and function of ceils. DNA replication is an essential process for the cell and ensures the accurate transmission of the cell type specific chromatin organization to the daughter cells. Moreoever, the chromatin pattern is different in normal and pathological cells. Although the analysis of specific aspects of the complex mechanisms involved have been assessed by means of analytical and quantitative cytology, the molecular processes that control chromatin replication is far from being understood. The majority of the studies have been done on fixed cells i.e. on cell snapshots and one should keep in mind that nuclear properties have been modified during the fixation steps and are thus different from nuclei of living cells. Experiments using microscopic imaging on living cells are necessary to clarify several aspects of the DNA replication. Microscopic imaging of living cells has been designed for structure-function analysis and can be considered, at the present, time, as the ultimate goal to understand the ph~,siology, biochemistry and biophysiscs of unperturbed, living cells. The objective of this method" is to study the cell functionnement (proliferation, differentiation and corresponding regulations) of living, normal and pathological cells, using fluorescent probes and image cytometry. Advances in probes design and staining as well as sample preparation techniques have provided a quantitative technology to measure some properties of living cells. A pilot study has been designed on MCF7 cells to determine : i) the experimental conditions to vitally and stoichiometrically stain the DNA (by Hoechst 33342, Ho 342) and ii) the secondary effects of Ho 342 on cell proliferation kinetics (using flow cytometry) for a further DNA content as well as chromatin pattern quantitation using image cytometry. The main objective is to compare the DNA content and the chromatin pattern in fixed and living cells.
c-fos proto-oncogene is a master switch converting short-term stimulations to long term-responses such as proliferation and differentiation. It is the cellular homolog of o n c o g e n e s carried by two t u m o r i g e n i c murine r e t r o v i r u s e s , FBR and FBJ. It encodes a s h o r t - l i v e d transcription,, f~.ctor interacting with the members of the jun f a m i l y into the API transcription c o m p l e x . Its regulation involves numerous transcriptional, posttranscriptional and post-translational levels of repression, the nmltiplicity of which likely explains why it has not yet been found constitutively expressed in natural tumors. We have recently pointed to the existence of two new gene activity regulation levels : (I) the transport of c-fos protein from the cytoplasm, where it is synthesized, into the nucleus, where it plays its part, is not constitutive but subjected to tight control by extracellular signals. (2) c-fos protein is extremely instable into the cytoplasm where its degradation is initiated by c a l c i u m - d e p e n d e n t proteases, a situation that departs from the nuclear one. I n t e r e s t i n g l y , FBR and FBJ viral proteins are c o n s t i t u t i v e l y transported into the nucleus and display lower sensitivity to c y t o p l a s m i c degradation, thus r e v e a l i n g two new m e c h a n i s m s contributing to their tumorigenic potential.
ALTERED HEAT-SHOCK RESPONSE IN MOUSE NIH 3T3 CELLS EXPRESSING EITHER v-FOS OR Ha-RAS 1 0 N C O G E N . FABRE. N.. DIAZ, C., MEHLEN, P., MAGAUD, J.P. (*) and A.P., ARRIGO.
Cloning of a human brain cDNA encoding a stimulatory GDP/GTP exchange protein for p21 Ha-ras. I.FATH, F. SCHWEIGHOFFER, M.C. CHEVALLIER-MULTON, M. FAURE**, I. BARLAT, F. RISBEC, M. J A C Q U E T * * , B. TOCQUE. RHONE POULENC RORER- 13 quai Jules Guesde- B.P. 14 94403 VITRY SUR SEINE cdde.x. FRANCE.** Groupe information g~ndtique et ddveloppement, URA 1354 CNRS, Universitd Paris Sud, 91405 ORSAY Cddex. FRANCE.
Molecular and Cellular GentHics, CNRS UMR-106, Claude Bernard University, Lyon-/, France. (°) HSpital Edouard Herriot, Lyon, France. Cancer cells are usually suceplible Io heat stress (1). Based on this properly, hyperlhermia is often used coupled with radiotherapy or chemotherapy in clinical Ireatments of superlicial lumors (2). To turlher investigate a putalive relationship between cell transformalion and heal sensitivity, we have analyzed the heal-shock response in mouse NIH 3T3 fibroblasts Iransformed with different oncogens, such as v-los and Ha-ras. We found Ihat both v-los and Ha-ras NIH 3T3 cells were more sensitive to hyperthermia than the isogenic unlransformed strain. At the protein level this phenomenon was characterised by a more drastic inhibition of translation of normal proleins at elevaled temperatures. Moreover, a decreased inducibility of the major heat-shock prolein hsp70 was observed in these cells. This effect was more pronounced in v-los 3T3 cells. Therefore, one putative function of hsp70 during heat stress, protection ol nucleolar pre-ribosomes, may be drastically impaired in these cancer cells. This effect may be specific to heal shock, since Ha-ras and v-los cells behave as normal 3T3 cells following exposure to hydrogen peroxide, a stress that only weakly induces hsp70. Our results support Ihe hypothesis thai Ihe hypersensitivily of some cancer cells Io hypertherrqia may be due to a decreased ability of these cells to Induce the molecular chaperone hsp70. This may lead to a lethal accumulation of misfolded proteins in these cells. Whether this decreased Inducibility of hsp70 occurs at the Iranscriplional or Iraduclional level ls currently under study. (1) HAHN, G.H., (1974). Cancer Res. 34, 3117-3123. (2) GERWECK, L.E., (1985). Cancer Res. 45, 3408-3414.
Activation of Ras p21 requires the presence of a g u a n i n e nucleotide exchange factor. This protein allows the dissociation of bound GDP and its replacement by GTP on Ras p21. Recently, Martegani et al. (Embo J.,1992, 11, 2151-2157) have isolated in mouse brain, a 1.2 kb partial cDNA, homologue of CDC25 (a g u a n i n e n u c l e o t i d e e x c h a n g e factor for RAS in y e a s t Saccharomyces cerevisiae) .
Based on this sequence, we screened a human brain stem library and isolated several full length cDNAs. A 3kb cDNA containing an open reading frame of 1,5 kb. encodes a protein of 50 kDa, s h a r i n g strong similarities with CDC25 and SOS (Son of Sevenless, a guanine nucleotide exchange factor for Drosophila R a s l protein). Northern blot analysis of h u m a n brain poiyA" RNA reveals one major transcript of 6 kb and a weaker signal of 3 kb. This cDNA seems to be brain specific as no transcript was found in other tissues such as liver, lung and placenta. The guanine nucleotide exchange activity of the C-term part of t h i s h u m a n b r a i n protein (278 last a m i n o acids) was demonstrated in vitro on human Ha-ras. This 278 amino acids protein was also found to complement the CDC25-5 ts. mutation inS. cerevisiae. Moreover, we have shown that the 3'-part of this h u m a n brain cDNA transactivates a ras responsive element in vivo in CHO cells.
C o l l o q u e "ContrSle M o l t c u l a i r e du C y c l e Cellulaire", Lyon, juillet 1992
MICIt, OSCOI'IC IMAGING OF T I l E CIIROMATIN IN LIVING CELLS - A P I L O T S T U D Y RONOT Xavier (1), COZ.ZIJean (2), GRUNrWALD Didier (3), ROBERT-NICOUD Michel (1), BRUGAL Gtrard (1), SELE Bernard (2). (I) Equipe de Reconnaissance des Formes et Microscopic Quantitative, Lab. "I1M3-1MAG, UniversitE J. Fourier, BP53X, F-38041 Grenoble Cedex. (2) Lab. de Biologie de la Reproduction, Facuh# de MEdecine. Domaine de la Merci , I:-38706 La Tronche Cedex. (3) DBMS, Lab. de Biologic Molt~culaire du Cycle Cellulaire, CENG, BP85X F-38041Grenoble Cedex.
CONTROL OF INTRACELLULAR LOCALIZATION D E G R A D A T I O N O F c-FOS P R O T E I N
AND
Seree C A R I L L O . Pierre ROUX, Ann-Muriel STEFF, Maryse E T I E N N E - J U L A N , J e a n - M a r i e B L A N C H A R D , Philippe JEANTEUR and Marc PIECHACZYK Laboratoire de Biologie Moldculaire. UA CNRS 1191 "GEndtique MolEculaire". place E. Baraillon -34095 - Montpellier Cede.r 05 France
One l~oal of cell biology is to explain interactions between molecules in a functtonal cell. The developments and applications of flow cytometry and fluorescence image cytometry have added new dimensions to knowledge about the structure and function of ceils. DNA replication is an essential process for the cell and ensures the accurate transmission of the cell type specific chromatin organization to the daughter cells. Moreoever, the chromatin pattern is different in normal and pathological cells. Although the analysis of specific aspects of the complex mechanisms involved have been assessed by means of analytical and quantitative cytology, the molecular processes that control chromatin replication is far from being understood. The majority of the studies have been done on fixed cells i.e. on cell snapshots and one should keep in mind that nuclear properties have been modified during the fixation steps and are thus different from nuclei of living cells. Experiments using microscopic imaging on living cells are necessary to clarify several aspects of the DNA replication. Microscopic imaging of living cells has been designed for structure-function analysis and can be considered, at the present, time, as the ultimate goal to understand the ph~,siology, biochemistry and biophysiscs of unperturbed, living cells. The objective of this method" is to study the cell functionnement (proliferation, differentiation and corresponding regulations) of living, normal and pathological cells, using fluorescent probes and image cytometry. Advances in probes design and staining as well as sample preparation techniques have provided a quantitative technology to measure some properties of living cells. A pilot study has been designed on MCF7 cells to determine : i) the experimental conditions to vitally and stoichiometrically stain the DNA (by Hoechst 33342, Ho 342) and ii) the secondary effects of Ho 342 on cell proliferation kinetics (using flow cytometry) for a further DNA content as well as chromatin pattern quantitation using image cytometry. The main objective is to compare the DNA content and the chromatin pattern in fixed and living cells.
c-fos proto-oncogene is a master switch converting short-term stimulations to long term-responses such as proliferation and differentiation. It is the cellular homolog of o n c o g e n e s carried by two t u m o r i g e n i c murine r e t r o v i r u s e s , FBR and FBJ. It encodes a s h o r t - l i v e d transcription,, f~.ctor interacting with the members of the jun f a m i l y into the API transcription c o m p l e x . Its regulation involves numerous transcriptional, posttranscriptional and post-translational levels of repression, the nmltiplicity of which likely explains why it has not yet been found constitutively expressed in natural tumors. We have recently pointed to the existence of two new gene activity regulation levels : (I) the transport of c-fos protein from the cytoplasm, where it is synthesized, into the nucleus, where it plays its part, is not constitutive but subjected to tight control by extracellular signals. (2) c-fos protein is extremely instable into the cytoplasm where its degradation is initiated by c a l c i u m - d e p e n d e n t proteases, a situation that departs from the nuclear one. I n t e r e s t i n g l y , FBR and FBJ viral proteins are c o n s t i t u t i v e l y transported into the nucleus and display lower sensitivity to c y t o p l a s m i c degradation, thus r e v e a l i n g two new m e c h a n i s m s contributing to their tumorigenic potential.
ALTERED HEAT-SHOCK RESPONSE IN MOUSE NIH 3T3 CELLS EXPRESSING EITHER v-FOS OR Ha-RAS 1 0 N C O G E N . FABRE. N.. DIAZ, C., MEHLEN, P., MAGAUD, J.P. (*) and A.P., ARRIGO.
Cloning of a human brain cDNA encoding a stimulatory GDP/GTP exchange protein for p21 Ha-ras. I.FATH, F. SCHWEIGHOFFER, M.C. CHEVALLIER-MULTON, M. FAURE**, I. BARLAT, F. RISBEC, M. J A C Q U E T * * , B. TOCQUE. RHONE POULENC RORER- 13 quai Jules Guesde- B.P. 14 94403 VITRY SUR SEINE cdde.x. FRANCE.** Groupe information g~ndtique et ddveloppement, URA 1354 CNRS, Universitd Paris Sud, 91405 ORSAY Cddex. FRANCE.
Molecular and Cellular GentHics, CNRS UMR-106, Claude Bernard University, Lyon-/, France. (°) HSpital Edouard Herriot, Lyon, France. Cancer cells are usually suceplible Io heat stress (1). Based on this properly, hyperlhermia is often used coupled with radiotherapy or chemotherapy in clinical Ireatments of superlicial lumors (2). To turlher investigate a putalive relationship between cell transformalion and heal sensitivity, we have analyzed the heal-shock response in mouse NIH 3T3 fibroblasts Iransformed with different oncogens, such as v-los and Ha-ras. We found Ihat both v-los and Ha-ras NIH 3T3 cells were more sensitive to hyperthermia than the isogenic unlransformed strain. At the protein level this phenomenon was characterised by a more drastic inhibition of translation of normal proleins at elevaled temperatures. Moreover, a decreased inducibility of the major heat-shock prolein hsp70 was observed in these cells. This effect was more pronounced in v-los 3T3 cells. Therefore, one putative function of hsp70 during heat stress, protection ol nucleolar pre-ribosomes, may be drastically impaired in these cancer cells. This effect may be specific to heal shock, since Ha-ras and v-los cells behave as normal 3T3 cells following exposure to hydrogen peroxide, a stress that only weakly induces hsp70. Our results support Ihe hypothesis thai Ihe hypersensitivily of some cancer cells Io hypertherrqia may be due to a decreased ability of these cells to Induce the molecular chaperone hsp70. This may lead to a lethal accumulation of misfolded proteins in these cells. Whether this decreased Inducibility of hsp70 occurs at the Iranscriplional or Iraduclional level ls currently under study. (1) HAHN, G.H., (1974). Cancer Res. 34, 3117-3123. (2) GERWECK, L.E., (1985). Cancer Res. 45, 3408-3414.
Activation of Ras p21 requires the presence of a g u a n i n e nucleotide exchange factor. This protein allows the dissociation of bound GDP and its replacement by GTP on Ras p21. Recently, Martegani et al. (Embo J.,1992, 11, 2151-2157) have isolated in mouse brain, a 1.2 kb partial cDNA, homologue of CDC25 (a g u a n i n e n u c l e o t i d e e x c h a n g e factor for RAS in y e a s t Saccharomyces cerevisiae) .
Based on this sequence, we screened a human brain stem library and isolated several full length cDNAs. A 3kb cDNA containing an open reading frame of 1,5 kb. encodes a protein of 50 kDa, s h a r i n g strong similarities with CDC25 and SOS (Son of Sevenless, a guanine nucleotide exchange factor for Drosophila R a s l protein). Northern blot analysis of h u m a n brain poiyA" RNA reveals one major transcript of 6 kb and a weaker signal of 3 kb. This cDNA seems to be brain specific as no transcript was found in other tissues such as liver, lung and placenta. The guanine nucleotide exchange activity of the C-term part of t h i s h u m a n b r a i n protein (278 last a m i n o acids) was demonstrated in vitro on human Ha-ras. This 278 amino acids protein was also found to complement the CDC25-5 ts. mutation inS. cerevisiae. Moreover, we have shown that the 3'-part of this h u m a n brain cDNA transactivates a ras responsive element in vivo in CHO cells.