Synchronization of human diploid fibroblasts at multiple stages of the cell cycle*1

Tobey, Robert A.; Valdez, J.G.; Crissman, Harry A.

doi:10.1016/0014-4827(88)90279-0

Cited by 73 publications

(53 citation statements)

References 22 publications

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“…HSF-55 diploid fibroblasts, derived by D. Chen (Los Alamos National Laboratory) from a human neonate foreskin sample, were grown as monolayers in 75-cm2 tissue culture flasks in a minimum essential medium supplemented with 10o heat-inactivated bovine calf serum and antibiotics (aMEM/lOC). Flow-cytometric analysis of DNA content in mithramycin-stained cells was done as described (15 (18), after which the monolayers were exposed to aMEM/lOC containing aphidicolin at 5 ttg/ml for 24 hr to arrest the cells in very-early-S phase (18). The (20,25).…”

Section: Methodsmentioning

confidence: 99%

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Cell cycle synchronization: reversible induction of G2 synchrony in cultured rodent and human diploid fibroblasts.

Tobey

Oishi

Crissman

1990

Proc. Natl. Acad. Sci. U.S.A.

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In accord with a set of prespecified principles of cell synchrony induction, a three-step procedure was developed to arrest cells reversibly in the G2 phase of the cell cycle. Cultures of Chinese hamster ovary (CHO) cells were presynchronized in early S phase by sequential treatment with isoleucine deficiency and hydroxyurea blockades; then they were switched to medium supplemented with either of two agents that inhibit DNA topoisomerase II activity by different mechanisms, Hoechst 33342 at 7.5 ,Lg/ml for 12 hr or VM-26 at 0.5 ,gg/ml for 8 hr. Up to 95% of the cells accumulated in G2 phase under those conditions. After switch of Hoechst 33342-treated cells to drug-free medium, the cells divided as a highly synchronized cohort of cells within 3 hr. Up to 85% of the cells in a culture of human diploid dermal fibroblasts (HSF-55 cells) could be accumulated in G2 phase by placing cells presynchronized in early-S phase in medium containing Hoechst 33342 at 0.1 jug/ml for 10 hr. Reversal of G2 arrest in the HSF-55 cultures resulted in cells dividing sychronously over 3.5 hr. By varying the concentration of Hoechst 33342 and the duration of the treatment period, it was possible to alter the position within G2 phase at which cells accumulated. This synchronization protocol should greatly facilitate study of G2/M biochemical events in mammalian cells, in particular, those associated with cdc2 gene regulation of the onset of mitosis.Studies of events within the mammalian cell cycle are greatly facilitated by the use of highly synchronized populations in which all or most cells perform the same biochemical operations simultaneously. Among factors to be considered when using or developing a synchronization protocol are the following: (i) avoidance of conditions that force cells into a state of "biochemical imbalance" (1) in which the ratios of major macromolecules are grossly perturbed; (ii) assurance that the synchronization protocol is completely reversible; and (iii) recognition that the synchrony is rapidly lost (2, 3) to the extent that no single synchronization protocol is suitable for study of events throughout the entire cell cycle.In this manuscript, these principles of synchrony induction have been applied to the development of a protocol to arrest mammalian cells reversibly in G2 phase. The need for such populations stems from the recent rekindling of interest in studies of the cell cycle, attributable to the development of techniques for elucidating the mechanisms responsible for genetic regulation of cell proliferation. In particular, studies with a variety of biological systems, predominantly involving the lower eukaryotes, have revealed the existence of a series of cdc2-like genes shown to play a crucial role in regulating the initiation of mitosis (4-11). These studies are currently focusing on the molecular interactions between the proteins encoded by the cdc2 gene and cellular constituents that trigger the transition from G2 phase into mitosis.Such studies previously were difficult to do with mamma...

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Section: Methodsmentioning

confidence: 99%

“…mented with aphidicolin (18). After additional exposure to medium containing 0.17 ,uM Hoechst 33342 at 0.1 ,ug/ml for 10 hr, populations containing 80-85% G2 cells were obtained (0-hr sample in Fig.…”

Section: Methodsmentioning

confidence: 99%

Cell cycle synchronization: reversible induction of G2 synchrony in cultured rodent and human diploid fibroblasts.

Tobey

Oishi

Crissman

1990

Proc. Natl. Acad. Sci. U.S.A.

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“…For synchrony in G 1 , cells were incubated in 0.5% FCS-containing medium for 48 h. For synchrony in S phase, cells were serum starved as described above for 48 h and then split into complete medium containing 1 mM hydroxyurea and incubated for 24 h (27). Cells were then released from hydroxyurea for 3 h, at which time most cells were in S phase.…”

Section: Cells and Mediamentioning

confidence: 99%

DNA Replication Is Required To Elicit Cellular Responses to Psoralen-Induced DNA Interstrand Cross-Links

Akkari

Bateman

Reifsteck

et al. 2000

Molecular and Cellular Biology

185

153

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Following introduction of DNA interstrand cross-links (ICLs), mammalian cells display chromosome breakage or cell cycle delay with a 4N DNA content. To further understand the nature of the delay, previously described as a G 2 /M arrest, we developed a protocol to generate ICLs during specific intervals of the cell cycle. Synchronous populations of G 1 , S, and G 2 cells were treated with photoactivated 4-hydroxymethyl-4,5,8-trimethylpsoralen (HMT) and scored for normal passage into mitosis. In contrast to what was found for ionizing radiation, ICLs introduced during G 2 did not result in a G 2 /M arrest, mitotic arrest, or chromosome breakage. Rather, subsequent passage through S phase was required to trigger both chromosome breakage and arrest in the next cell cycle. Similarly, ICLs introduced during G 1 did not cause a G 1 /S arrest. We conclude that DNA replication is required to elicit the cellular responses of cell cycle arrest and genomic instability after psoralen-induced ICLs. In primary human fibroblasts, the 4N DNA content cell cycle arrest triggered by ICLs was long lasting but reversible. Kinetic analysis suggested that these cells could remove up to ϳ2,500 ICLs/genome at an average rate of 11 ICLs/genome/h.Many clinically important chemotherapeutic chemicals can induce DNA interstrand cross-links (ICLs). These include mitomycin C (MMC), diepoxybutane, nitrogen and sulfur mustards, cisplatin, and photoactivated psoralens (21). ICLs pose a particular challenge to DNA repair systems since they involve both strands of DNA and cannot, therefore, be repaired using the redundant information in the complementary strand.In Escherichia coli and yeast, the repair of ICLs involves the sequential action of several repair pathways. In E. coli, genetic and biochemical studies pointed to a recombinational-incisional repair (4, 13) in addition to a pathway involving a DNA glycosylase (28). In yeast, unlike in E. coli, double-strand breaks occur in response to ICLs. The repair of these breaks is dependent on the presence of RAD52 (homologous recombination repair) and RAD2 (excision repair) but not on RAD6 (mutagenic repair) (5,12,19).Much less is known about the biology of cross-link repair in mammalian cells. Many of the studies in this field have focused on Fanconi anemia (FA) cells because of their sensitivity to cross-linking agents. The FA cellular phenotype is manifested as increased chromosome breakage (1) and a marked cell cycle delay with a 4N DNA content after introduction of ICLs (16). This delay has been also described as a G 2 /M checkpoint. It has therefore been suggested that the molecular basis of FA is a defect in the repair of ICLs (25), but to date no direct support for this hypothesis has been presented. After treatment with agents that induce ICLs, wild-type cells also display cell cycle delay with 4N DNA content and chromosome breakage, but the doses required are higher (10,14).In this study, we set out to determine the nature of the cell cycle delay with 4N DNA content in response to ICLs in wi...

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“…for 24 to 27.5 h (16,57). In one experiment, cells treated with hydroxyurea were washed three times with phosphate-buffered saline (PBS) to release the cells from drug arrest and then overlaid again with fresh media.…”

mentioning

confidence: 99%

“…Cultures incubated in the presence of hydroxyurea or nocodazole for 20.5 h were washed twice with PBS and starved in methionineand cysteine-free medium containing the appropriate drugs and 5% fetal calf serum for 3.5 h (16,57).…”

mentioning

confidence: 99%

Identification of a rel-related protein in the nucleus during the S phase of the cell cycle.

Evans¹,

Gottlieb²,

Bose³

1993

Mol. Cell. Biol.

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The c-rel proto-oncogene encodes a 75-kDa protein (p75cI) which is present in the cytosol of chick embryo fibroblasts (CEF) associated with a distinct set of cellular proteins with molecular masses of 40, 115, and 124 kDa. CEF cultures arrested in S phase of the cell cycle, or enriched for G2 or mitotic cells, were examined to determine whether the expression of c-rel was altered during the cell cycle. Levels of p75C`remained constant in all portions of the cell cycle examined; however, a Rel-related protein with an apparent molecular mass of 64 kDa was detected in nuclei of S-phase cells. As cells enter G2, the level of this protein in the nucleus decreases. This protein reacts with antiserum generated against the carboxy terminus of p75C" in radioimmunoprecipitations and Western immunoblot experiments and was also detected in a Western immunoblot with antiserum generated against the first 161 amino acids of pp59v". Thus, unlike other Rel/NF-KB family members, p64 has carboxy-terminal homology with c-Rel. The majority of peptides generated by partial proteolytic cleavage of p64 are shared with peptides generated by digestion of p75C and/or pp59v'. We suggest that this protein represents a new member of the Rel family of transcription factors and is located in the nucleus of avian fibroblasts during S phase of the cell cycle.The proto-oncogene c-rel encodes a 75-kDa protein (p75C'-) which is a member of the Rel/NF-KB family of transcription factors (38,53). This family includes the product of the v-rel oncogene carried by reticuloendotheliosis virus (REV-T), the Drosophila melanogaster maternal morphogen Dorsal, the various subunits of the mammalian transcription complex NF-KB, human p49, and murine RelB and its human homolog I-Rel (24,34,42,(46)(47)(48)55). These proteins share homology within approximately 350 amino acids near the amino terminus (the Rel domain), and all except v-Rel and c-Rel are completely divergent at their carboxy termini (10,34,42,47,48). With one exception, these proteins are able to bind DNA in vitro to the consensus NF-KB binding site (KB site) as homo-or heterodimers (4,6,34,42,44,47,48). Dorsal has not been shown to form heterodimers, nor has it been shown to bind the consensus KB site; however, it does bind a related site in the zen promoter (31). Recent data utilizing degenerate oligonucleotides indicate that different combinations of Rel family members preferentially bind divergent KB-related motifs (44). Which Rel family members heterodimerize in vivo and are physiologically active is still a matter of speculation.NF-KB was initially identified in lymphoid cells as a heterodimer composed of a 50-kDa subunit (p50) which is derived from a 110-kDa precursor by proteolytic cleavage and a 65-kDa subunit (p65) (2-4, 23, 25, 34, 58). This DNA-binding complex is sequestered in the cytosol by association with the inhibitory protein IKB (2, 3). p75C"' is found in the cytosol of avian lymphocytes associated with three major proteins (40, 115, and 124 kDa) (15,19,35,40). The 40-kDa protein ...

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Synchronization of human diploid fibroblasts at multiple stages of the cell cycle*1

Cited by 73 publications

References 22 publications

Cell cycle synchronization: reversible induction of G2 synchrony in cultured rodent and human diploid fibroblasts.

Cell cycle synchronization: reversible induction of G2 synchrony in cultured rodent and human diploid fibroblasts.

DNA Replication Is Required To Elicit Cellular Responses to Psoralen-Induced DNA Interstrand Cross-Links

Identification of a rel-related protein in the nucleus during the S phase of the cell cycle.

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