Chinese Hamster Embryo Fibroblast Cells Research Articles

Flow karyotype analysis and sorting of the Chinese hamster chromosomes: Comparing the effects of the isolation buffers

The effects of different swelling solutions on the univariate flow karyotype of whole chinese hamster embryo cells (WCHE K10) and cloned chinese hamster embryo fibroblast cells (CCHE 40) were compared using four methods of chromosome isolation. Chromosomes of each cell line were prepared by the Aten, Polyamine-Digitonin, Tris-Triton, and HEPES methods and analyzed with a FACStar flow cytometer. Polyamine-Digitonin and Aten methods produced the most satisfactory flow karyotype. Structurally aberrant chromosomes in the cell lines were detected in the flow karyotype as extra peaks. CCHE 40 chromosomes 1,2,X and 3q were sorted into separate tubes and reanalyzed; and the coefficient of variation of each chromosome peak was near 5%. To apply the flow karyotype analysis to clinical cytogenetics, chromosome preparation was one of the most important factor, and it is necessary to sort the chromosome peak sharply.

Calmodulin-specific monoclonal antibodies inhibit DNA replication in mammalian cells.

The involvement of calmodulin in the proliferation of Chinese hamster embryo fibroblast cells has been studied with a specific monoclonal antibody to calmodulin. We observed that calmodulin levels increase 2-fold in the late G1 period in these cells, and this coincides with the increase in DNA polymerase alpha activity as the cells progress synchronously from a quiescent state in the G1 to the S phase. However, there is a concurrent 10-fold enhancement of thymidine kinase activity, which is tightly coupled to the entry of cells into the S phase. Incubation of permeabilized S-phase cells with calmodulin-specific murine monoclonal antibody resulted in a dose-dependent inhibition of DNA replication. This inhibitory effect of anti-calmodulin antibodies on DNA replication is completely reversed by the addition of exogenously purified calmodulin. These observations provide evidence for the involvement of calmodulin in DNA replication and, therefore, in cell proliferation during the S phase.

Nuclear localization of 68 kDa calmodulin‐binding protein is associated with the onset of DNA replication

In Chinese hamster embryo fibroblast cells, an increase in intracellular calmodulin levels coincided with the nuclear localization of a calmodulin-binding protein of about 68 kDa as the cells progressed from G1 to S phase. When cells were limited from entering into S phase, by omitting insulin a defined medium, intracellular CaM levels did not increase and the 68 kDa calmodulin-binding protein was completely absent from the nuclei. Corresponding to the nuclear localization of calmodulin and the 68 kDa calmodulin-binding protein in S phase cells, there was a dramatic increase in DNA polymerase and thymidine kinase activities in the nuclei of S phase cells as compared to G1 phase cells. In addition, the 68 kDa calmodulin-binding protein, along with calmodulin, is observed to be an integral component of replitase complex responsible for nuclear DNA replication in S phase cells. These observations point to the association of calmodulin and calmodulin-binding protein(s) with the replication machinery responsible for nuclear DNA replication during S phase. A possible regulatory role of these proteins in the onset of DNA replication and cell proliferation is discussed.

Compartmentation of deoxypyrimidine nucleotides for nuclear DNA replication in S phase mammalian cells.

DNA synthesis in S phase Chinese hamster embryo fibroblast cells in the presence of exogenous 3H-dUrd shows incorporation of the labeled precursor with very little dilution by the large unlabeled intracellular precursor pools. Full mixing would predict a specific activity 10-fold less than that measured. This coupled with the finding that 80% of the radioactivity derived from the exogenous 3H-dUrd appears in the karyoplasts implies a compartmentation where 3H-dUMP and 3H-dTTP derived from exogenous 3H-dUrd do not mix freely with endogenous cytoplasmic pools.

Association of 3′ → 5′ exodeoxyribonuclease activity with DNA replitase complex from S-phase Chinese hamster embryo fibroblast cells

DNA replitase has been described as a complex of enzymes/proteins that are associated with both DNA precursor biosynthesis and DNA replication in mammalian cells [ Reddy, G. P. V., and Pardee, A. B. (1980) Proc. Natl. Acad. Sci. USA 77, 3312–3316]. We demonstrate for the first time a 3′ → 5′ exodeoxyribonuclease activity is associated with the replitase complex. As much as 60% of this exonuclease activity was similar to that associated with DNA polymerase δ based upon its sensitivity to inhibition by GMP and by butyl-phenyl-deoxyguanosine triphosphate (BuPdGTP). Association of 3′ → 5′ exonuclease activity with the DNA polymerase in the replitase complex was also demonstrated by analyzing dTTP turnover to dTMP in an in vitro DNA polymerase assay system. The DNA polymerase activity in replitase complex exhibited a sensitivity to BuPdGTP which both was similar to that of DNA replication in permeable cells and was intermediate between the BuPdGTP inhibition of purified DNA polymerases α and δ. These studies suggest that the replitase complex contains 3′ → 5′ exonuclease activity associated with the DNA polymerase activity responsible for nuclear DNA replication in mammalian cells. Further studies are required to determine if these activities are at least partially attributed to DNA polymerase δ.

Both protein kinase C and calcium mediate activation of the Na+/H+ antiporter in Chinese hamster embryo fibroblasts.

Chinese hamster embryo fibroblast cells (CHEF/18) possess a plasma membrane-associated, amiloride-sensitive Na+/H+ antiporter that affects intracellular pH (pHi) and is activated by growth factor addition. Our results using 14C-benzoic acid distribution indicate that both epidermal growth factor (EGF) and thrombin are capable of causing rapid rises in the pHi of CHEF/18 cells. The maximal shift induced by these factors is 0.20 to 0.25 pH units above the basal unstimulated level. Distinctive differences were observed between the modes of action of these two growth factors. Sequential additions revealed that the rise in pHi due to EGF was additive with that caused by diacylglycerols (DAG), while that of thrombin was not. Furthermore, exposure of cells to the phorbol ester PMA for a prolonged period of time in order to down-regulate protein kinase C (pkC), or treatment with the pkC inhibitor H-7, abolished the pHi response to thrombin but not to EGF. In contrast, incubation of cells in nominally calcium-free medium or with the calmodulin antagonists W-7 or trifluoperazine (TFP) decreased only the ability of EGF to cause changes in pHi. These data suggest that there are two distinct mechanisms for activation of the Na+/H+ antiporter in CHEF/18 fibroblast cells and thus provide an example of the use of alternative modes for the modulation of intracellular processes.

Intracellular pH is increased after transformation of Chinese hamster embryo fibroblasts.

These studies reveal that a series of tumorigenic Chinese hamster embryo fibroblast (CHEF) cell lines maintain an internal pH (pHi) that is 0.12 +/- 0.04 pH unit above that of the nontumorigenic CHEF/18 parental line. This increase of pHi in the tumorigenic CHEF cells is not due to autocrine growth factor production or to the persistent activation of pathways previously shown to modulate Na+/H+-antiporter activity present in the CHEF/18 line. These findings suggest that the defect in pHi regulation in the tumorigenic CHEF/18 derivatives lies in the Na+/H+ antiporter itself. Further studies to determine the biological significance of an increased pHi show that the external pH (pHo)-dependence curve for initiation of DNA synthesis in the tumorigenic CHEF lines is shifted by approximately 0.2 pH unit toward acidic values relative to that of the nontumorigenic CHEF/18 parent. These data show a critical role for pHi in the regulation of DNA synthesis in Chinese hamster embryo fibroblasts and demonstrate that aberrations in pHi can contribute to the acquisition of altered growth properties.

Plasmid-induced "hit-and-run" tumorigenesis in Chinese hamster embryo fibroblast (CHEF) cells.

The Chinese hamster embryo fibroblast cell line CHEF/18 is readily transfected by plasmid DNA. In the present transfection studies with CHEF/18 cells, focus formation induced by plasmids containing the mutant human c-Ha-ras gene EJ was compared with that of control plasmids without the EJ insert. The focus-forming activity of the transfected plasmid J132, a recombinant of the Harvey murine sarcoma virus LTR and the normal human c-Ha-ras1 in pBR322, also was assessed. Foci were recovered after transfection with either pSV2gpt or pSV2neo at about 10% the frequency obtained with the EJ-containing plasmids, and J132 gave a similar frequency, all well above background obtained with salmon sperm DNA. Whereas foci from transfection with EJ-containing plasmids contained the EJ DNA, no plasmid DNA was detected in either tumorigenic or tumor-derived cells from foci transfected with pSVgpt, pSVneo, or J132. Evidence that genomic changes were induced by plasmid transfection is based on finding chromosomal aberrations in all expanded foci and tumor-derived cells examined. The results suggest the occurrence of "hit-and-run" tumorigenesis induced by transient plasmid transfection.

Suppression of tumorigenicity in hybrids of normal and oncogene-transformed CHEF cells.

Somatic cell hybridization experiments were carried out to determine whether normal cells have the ability to suppress the transforming effects of a defined oncogene. A nontransformed Chinese hamster embryo fibroblast cell line (CHEF/18-dm2) was used as the normal parent, and a CHEF/18 transfectant carrying the human mutant c-Ha-ras (EJ) oncogene was used as the tumorigenic parent. Selected hybrids (L318 cell lines) were assayed for the presence of EJ DNA, for the p21 product of the c-Ha-ras gene, and for various indices of cell transformation. These hybrids exhibited a fibroblastic morphology similar to the normal parent, although they contained the EJ gene and expressed its p21 protein product at levels comparable with the transformed parent. They had a reduced capacity for anchorage-independent growth (plating efficiency in methylcellulose of less than 0.3-13%, as compared with greater than 90% for the transformed parent) and decreased tumor-forming ability in athymic mice. These findings show that normal CHEF/18 cells contain suppressor genes capable of inhibiting expression of the transformed phenotype, and tumor-forming ability, in the presence of an activated EJ oncogene.

Genetic analysis of tumorigenesis: XVI. Chromosome changes in azacytidine- and insulin-induced tumorigenesis.

Chromosome changes accompanying differentiation and tumorigenesis in azacytidine- (azaC) and insulin-induced preadipocytes of the Chinese hamster embryo fibroblast cell line CHEF/18 are described. Karyotype analysis of 47 clones, subclones, and tumor-derived cells has shown that trisomy for chromosome 3q (mar 1) is characteristic of azaC preadipocytes but not of insulin preadipocytes. AzaC preadipocytes were consistently tumorigenic as well as trisomic for chromosome 3q, whereas most insulin preadipocytes were nontumorigenic and diploid. Only the few insulin preadipocytes that were tumorigenic were also trisomic for chromosome 3q. Among the tumor-derived cell lines recovered from azaC preadipocytes injected into nude mice, four had no additional chromosome changes except trisomy for 3q, as detected by karyotype analysis. Thus trisomy for 3q may be a sufficient chromosome change to induce tumor-forming ability in these cells. The rearrangements of chromosome 3 seen in this and other work pinpoint the trisomic region between the centromere and 3q5.

Catalytic function of thymidylate synthase is confined to S phase due to its association with replitase

Catalytic activity of thymidylate synthase, as measured in, vivo , is tightly linked to S phase of the cell cycle in Chinese hamster embryo fibroblast cells. This activity, as measured in, vitro , is found in all parts of the cell cycle. Thymidylate synthase activity in nuclear (karyoplast) extracts increased as the cells progressed from G 0 G 1 to S phase. This enzymatic activity in the nuclei of S phase cells is associated with the multienzyme complex (replitase) that also contained DNA polymerase and other enzymes of DNA replication and precursor synthesis. The degree of association of thymidylate synthase with replitase, which increased co-ordinately as the cells progressed from G 0 G 1 phase to S phase, coincided strongly with the level of in, vivo activity of the enzyme.

Genetic analysis of tumorigenesis: X. Chromosome studies of transformed mutants and tumor-derived CHEF/18 cells.

Chinese hamster embryo fibroblast cell line CHEF/18 is stably diploid, anchorage-dependent, has a high serum requirement, and a does not form tumors in nude mice. The chromosome constitutions of spontaneous and chemically induced anchorage-independent and/or low-serum CHEF/18 mutants and tumors produced in nude mice by some of these mutants are compared. We find a correlation between diploidy and nontumorigenicity among the anchorage-independent mutants but not in the low-serum mutants. One of the four spontaneous and six of the 15 chemically induced anchorage mutants have remained diploid. The remaining 12 mutants are pseudodiploid or aneuploid, and seven of them contain changes in chromosome 1, either a translocation or a deletion involving breakage at the same position (1q11-12). Each of the tumors induced by six mutants has a unique pattern of rearrangements; however five of the six have changes involving chromosome 3. This chromosome was also frequency rearranged in tumor-derived cells previously investigated.

Multienzyme complex for metabolic channeling in mammalian DNA replication.

In the DNA-synthesizing phase (S phase) of CHEF/18 Chinese hamster embryo fibroblast cells, six enzymes associated with DNA metabolism, including DNA polymerase (deoxynucleoside triphosphate:DNA deoxynucleotidyl-transferase, EC 2.7.7.7), were largely localized in the nuclear region (karyoplasts). By contrast, in quiescent and G1 phase cells these enzymatic activites were mainly absent from the nucleus and were recovered in the cytoplasmic portion (cytoplasts). These nuclear (but not cytoplasmic) enzymatic activities cosedimented rapidly on sucrose density gradients. Further, the rapidly sedimenting enzyme activities were unique to cells in S phase. An organized supramolecular structure that allows channeling of metabolites into DNA was demonstrated by kinetics of nucleotide incorporation. "Permeabilized" cells selectively channeled incorporation of ribonucleoside diphosphates into DNA in preference to deoxyribonucleoside triphosphates. Deoxyribonucleoside triphosphate incorporation occurred when ribonucleoside-diphosphate reductase (2'-deoxyribonucleoside-diphosphate: oxidized-thioredoxin 2'-oxidoreductase, EC 1.17.4.1) activity was abolished by hydroxyurea. Our interpretation is that during DNA replication, the nucleus contains a complex of DNA precursor-synthesizing enzymes juxtaposed with the "replication apparatus" comprising DNA polymerase, other enzymes, and structural proteins. Functional integrity of this structure is impaired when one of its essential components is inactivated. We propose the name "replitase" for this multienzyme complex for DNA replication and suggest that it incorporates precursors rapidly and efficiently. Possibly its assembly signals the initiation of the S phase of the cell cycle.