High-passage Cells Research Articles

Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.

Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma.

Resistance of primary isolates of human immunodeficiency virus type 1 to neutralization by soluble CD4 is not due to lower affinity with the viral envelope glycoprotein gp120.

Recombinant soluble CD4 (rsCD4) has potent antiviral activity against cell line-adapted isolates of the human immunodeficiency virus type 1 (HIV-1) but low activity toward HIV-1 primary isolates from patients. A simple hypothesis proposed to explain this discrepancy, which questions the therapeutic utility of soluble CD4-based approaches, is that the major envelope glycoprotein, gp120, of patient virus has lower affinity for CD4 than does gp120 from laboratory viruses. To test this hypothesis, we have produced pairs of low- and high-passage HIV-1 isolates which, depending on culture passage history, display dramatically different sensitivities to neutralization by rsCD4. Here, we present evidence that the HIV-1 major envelope glycoprotein cDNAs cloned from one such isolate pair show only minor differences in their deduced gp120 primary structures, and these occur outside regions previously shown to be involved in CD4 interactions. In addition, recombinant gp120 from a low-passage rsCD4-resistant patient virus binds rsCD4 with high affinity, equal to that previously measured for recombinant gp120 from high-passage cell line-adapted virus isolates. These data indicate that differences in CD4-gp120 affinity do not account for rsCD4 resistance in HIV-1 recently isolated from patients.

Expression of carbohydrate binding protein 35 in human fibroblasts: Variations in the levels of mRNA, protein, and isoelectric species as a function of replicative competence

We have compared the expression and localization of carbohydrate binding protein 35 (CBP35) in human SL66 fibroblasts of different replicative capacities. When serum-starved, quiescent young (passage 11, corresponding to ~18 cumulative population doublings) SL66 cells were treated with serum, there was a marked stimulation in the expression of CBP35. This was revealed both by an increase in the percentage of cells positively stained with anti-CBP35 under immunofluorescence and by an increase in the amount of the protein in immunoblots, as well as by an increase in the level of accumulated mRNA in Northern blots. The rise in the expression of CBP35 in proliferating cells was manifested most clearly in the nuclear fraction, with elevation in the levels of the nonphosphorylated (p I 8.7) protein, as well as the phosphorylated (p I 8.2) derivative. In contrast, older (passage 27–35, 55–68 cumulative population doublings) cultures of SL66 fibroblasts appear to have lost the normal proliferation-dependent regulation of CBP35 expression. The level of CBP35 was high in quiescent high-passage cells and decreased somewhat after serum stimulation. Furthermore, the unphosphorylated (p I 8.7) form of the lectin could not be detected in either the nucleus or the cytoplasm of high-passage SL66 cells. Finally, the level of the mRNA for CBP35 was high in quiescent cultures of high-passage cells, but undetectable 17 h after serum stimulation. These results establish that the expression of CBP35 becomes altered as human fibroblasts acquire reduced replicative capacity.

Modulation of Glucose-Induced Insulin Secretion from a Rat Clonal β-Cell Line*

The present studies demonstrate that the beta-cell line RINr1046-38 (RIN-38) retains the capability to secrete insulin in response to glucose. The maximal effect of glucose was a 5- to 9-fold stimulation of insulin secretion from RIN-38 cells. This glucose-induced insulin secretion was maximal at 0.6 mM and was modulated by other secretagogues. Potassium concentrations of 10 mM, adenylate cyclase activators (glucagon-like peptide-1 and forskolin), and a phosphodiesterase inhibitor (isobutylmethylxanthine) potentiated glucose-induced insulin secretion, but had little or no effect on insulin secretion in the absence of glucose. Potassium concentrations of 20 mM or more, glibenclamide, and carbachol (Cch) stimulated insulin secretion 8- to 12-fold in the absence of glucose, while only Cch potentiated the effect of glucose on insulin secretion. Amino acids (alanine, arginine, leucine, and ketoisocaproate) also stimulated insulin secretion. The alpha 2-adrenergic agonist clonidine (1 microM), low extracellular calcium (less than or equal to 0.5 mM), and extended culture of RIN-38 cells at low glucose concentrations (0.33 mM) inhibited the stimulatory effect of glucose on insulin secretion. Insulin secretion was retained in RIN-38 cells for up to 98 passages. However, extended passage was associated with a decline in cellular insulin content (83% decline over 89 passages). In addition, high passage cells lost the ability to secrete insulin in response to glucose, but continued to respond to other secretagogues (K+, alanine, and carbachol). In fact, in the absence of glucose the effect of Cch on insulin secretion was well maintained in high passage cells (8- and 9.9-fold increase in insulin secretion, passages 9 and 70, respectively). Thus, low passage RIN-38 cells secrete insulin in response to glucose and other insulin secretagogues. High passage cells do not respond to glucose, but continue to respond to other secretagogues. Based on these results we propose that high and low passage RIN-38 cells provide a model for examining molecular mechanisms of glucose-induced insulin secretion. In addition, these findings emphasize that passage information is essential for interpretation of secretion studies with RIN cell lines.

Dimethylformamide-Induced Changes in the Radiation Survival of Low- and High-Passage Intestinal Epithelial Cells (IEC-17) in Vitro

The effects of dimethylformamide (DMF) on the radiation response of low- and high-passage intestinal epithelial cells (IEC-17) were examined. The IEC-17 cell line, a rat intestinal epithelial cell line, exhibited a bimodal response to X radiation. The sensitive fraction, which was attributed to a stem cell-like component, had a D0 of 0.90 Gy. The resistant fraction, thought to be the expression of a more mature component, exhibited a D0 of 2.00 Gy. Treatment using a putative cell differentiating agent, N,N-dimethylformamide, increased the resistant fraction of the population from 35 to 80%, suggesting that DMF treatment (100 mM) increased the proportion of mature cells in the IEC-17 cell population. In addition, extended age in culture (greater than 100 passages) resulted in altered morphology, decreased doubling time, increased chromosome number, and loss of anchorage dependence, all features characterizing spontaneously transformed high-passage IEC-17 cells. These high-passage cells also exhibited a bimodal response to X radiation; the sensitive fraction had a D0 of 0.80 Gy while the resistant fraction D0 was 1.50 Gy. DMF increased the resistant fraction from 35 to 55% of the population. Results suggested that the different radiosensitivities of the subpopulations remained throughout the spontaneous transformation of high-passage IEC-17 cells.

Studies of phospholipase and cyclooxygenase activities in herpes simplex virus type 2-transformed rat cells

In this report we describe results showing that both arachidonic acid (20:4) release and prostaglandin (PG) synthesis in herpes simplex virus type 2 (HSV-2)-transformed tumor-derived rat fibrosarcoma (RFS) cells are inhibited by indomethacin (cyclooxygenase inhibitor). These data suggest that cyclooxygenase (prostaglandin endoperoxide synthetase, PES) and phospholipase are coupled in their regulation in RFS cells. Data obtained using a cell-free assay to measure directly cyclooxygenase specific activity in the absence of phospholipase activity indicate that constitutive cyclooxygenase activity is very low in non-transformed REF and is not induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, after an initial degradation of constitutive enzyme, cyclooxygenase is synthesized de novo following TPA treatment of HSV-2-transformed RFS clonal cells. At least a 5-fold induction of cyclooxygenase occurs by 6 h post-treatment of HSV-2-transformed RFS clonal cells. This induction is inhibited by pretreatment of cells with cycloheximide (protein synthesis inhibitor) 1 h prior to TPA treatment. Results obtained following delayed addition of cycloheximide to cells after TPA treatment indicate that the newly synthesized cyclooxygenase has a half-life of approximately 5 min. In addition, HSV-2-transformed RFS clonal cells at low passage (less than 10) show over a 3-fold greater increase in cyclooxygenase specific activity after TPA treatment compared to that in TPA-treated high passage (greater than 20) cells, suggesting that induction of high levels of PG synthesis is transient in these cells. These data indicate that HSV-2 transformation of REF cells is associated with alteration of cyclooxygenase regulation.

Autologous antibodies to human bladder cancer.

The autologous serologic reactivity of 13 patients with bladder cancer was evaluated using cell lines derived from each individual's own tumor as targets. Protein A and immune adherence assays were employed to determine antibody binding to the tumor targets at varying passage numbers. Autologous reactivity was found in 6 of the 13 cell lines tested. However, the titer was usually low regardless of the passage number. Seven autologous serum/cell line combinations were tested using both low and high passage cells as targets. In six of these combinations, the degree of antibody binding was similar with both low and high passage target cells. The incidence of autologous reactivity in the 12 patients with urothelial tumors was 50%.

Instability of V-SRC sequences in nonhuman primate tumors cultured in vitro

We have analyzed the DNA of marmoset tumors induced and marmoset cells transformed by Rous sarcoma virus (RSV) and derivative viruses of various types. Southern blot hybridization was used to determine the presence of v- src gene sequences. We failed to detect v- src DNA in high-passage cells derived from marmoset tumors induced in vivo or from marmoset cell lines transformed in vitro. The inability to detect src sequences was not related to selection of revertants in culture, since all cell lines retained transformed morphology and cells transformed in vitro retained the ability to induce sarcomas after transplantation into adult allogeneic marmosets. By contrast, we detected integrated proviruses in cells analyzed 32 to 60 days after in vitro transformation. The proviral sequences appeared to be identical to the transforming virus but were apparently unstable and continued to transpose.

Immunocytochemical characterization of the A15 A5 transplantable brain tumour model in vivo.

A comparative immunocytochemical study was carried out on intracerebral and extracranial gliomas of the rat produced by intracerebral injection of low (10th) and high (40th) in vitro passages of neoplastic glial cells. The cells injected were a neoplastic astrocytic clone-A15 A5-derived from a mixed glioma induced transplacentally by N-ethyl-N-nitrosourea (ENU) in a BD-IX rat. An inverse relationship was seen between the expression of the astrocytic markers glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) at low and high passage: GFAP decreased with increasing passage while GS increased. The distribution of vimentin, the major cytoskeletal component of immature glia, was constant, irrespective of passage--a feature consistent with previous in vitro findings. The expression of laminin by both reactive and neoplastic astrocytes increased with increasing passage, while high magnification examination revealed the presence of the glycoprotein fibronectin on the cell-surfaces of A15 A5-derived tumour cells. Both neoplastic and reactive astrocytes expressed S-100 protein with a higher proportion of positive cells in extracranial tumours. Occasional cells, probably actively phagocytizing populations of reactive astrocytes and macrophages, were positive for alpha-1-antitrypsin. None of the neoplastic cells expressed the oligodendrocyte marker carbonic anhydrase II. This immunocytochemical study supports previous morphological findings in differences in differentiation between the cells of tumours produced by high and low passage cells.

The morphogenesis of yaba monkey tumor virus in a cynomolgus monkey kidney cell line

The morphogenesis of Yaba monkey tumor virus (YMTV) was investigated in a cynomolgus monkey kidney cell line. YMTV has been shown to grow less rapidly in high-passage cells (i.e., 65 passages) than in low-passage cells (i.e., between 30 and 60 passages). This prolonged growth cycle in high-passage cells allows for a more detailed analysis of the events in morphogenesis. Samples of YMTV-infected cells were prepared for analysis at 2, 3, 4, and 5 days postinfection. At least 12 "stages" of virus morphogenesis can be identified.

Characterization of a lung epithelial cell strain with potential applications in toxicological studies

An epithelial cell strain (LEC, lung epithelial cells) was established from the lungs of an adult Fischer-344 rat. The cells had properties of normal, untransformed cells in culture which include contact inhibition of growth, inability to grow in soft agar, and inability to form tumors when injected subcutaneously into syngeneic hosts. Low passage LEC were diploid, while high passage cells were aneuploid with an extra chromosome. LEC resembled type II alveolar cell in vivo in that they had lamellar inclusions in their cytoplasm, microvilli on cell surface, and that they could incorporate [ 14C] choline into disaturated phosphatidyl choline, a surfactant lipid. The ability of LEC to activate procarcinogens was demonstrated by co-cultivation of LEC with Chinese hamster ovary (CHO) cells in the presence of procarcinogens benzo[ a]pyrene and 3-methyl cholanthrene. While these procarcinogens were non-mutagenic to CHO cells which lacked the metabolic activation capacity, they were mutagenic when LEC were co-cultured with the CHO cells. By maintaining LEC cells on collagen gel without overlying medium, the cells could be directly exposed to noxious airborne agents. Using this technique, the cytotoxicity of NO 2 was detected. Because of the lung epithelial origin, the ability to metabolize xenobiotics, and the exposure system to airborne agents, the LEC strain can be a useful and relevant in vitro system for the toxicological studies of inhalable materials.

Regulation of α-fetoprotein and transferrin synthesis and secretion in mouse hepatoma cells

Significant differences in the glucocorticoid- and cyclic nucleotide-mediated regulation of the secretory glycoproteins, α-fetoprotein and transferrin, have been observed to develop in a mouse hepatoma cell line, Hepa-2, after many passages in culture. Treatment of low-passage cells with hydrocortisone (10 −6 m), N 6,O 2 -dibutyryl cyclic AMP (10 −3 m), or 8-bromo-cyclic AMP (10 −3 m) results in 1.5-, 2- to 4-, and 5.5- to 6-fold increases, respectively, in the rates of synthesis and secretion of α-fetoprotein. As expected of secretory proteins, the ratio of synthesis to secretion is 1 and remains unaltered when treatment with hydrocoritsone, N 6,O 2 -dibutyryl cyclic AMP, and 8-bromo-cyclic AMP causes a stimulation of synthesis and secretion. Similar studies showing that albumin and transferrin synthesis and secretion are also balanced in these low-passage cells have been published and indicate that the regulation of synthesis and secretion remains coupled in these low-passage cells. In high-passage Hepa-2 cells, however, we have shown that the relative rate of α-fetoprotein synthesis is higher than its rate of secretion and that the ratio of synthesis to secretion is 4. Similarly, the ratio of transferrin synthesis to secretion is 3.6, whereas it remains unaltered for albumin. When the high-passage cells are treated with N 6,O 2 -dibutyryl cyclic AMP, there is a greater increase in the rate of secretion for both glycoproteins, resulting in a reduction of the ratio of synthesis to secretion from 4 to 1.63 for α-fetoprotein and from 3.6 to 2.3 for transferrin. This effect on the secretion of α-fetoprotein and transferrin is specific for the cyclic nucleotides and occurs only in high-passage cells. Hydrocortisone treatment causes an increase in α-fetoprotein synthesis and secretion. However, the ratio of synthesis to secretion increases from 3.96 in control to 5.5 in treated cells. Our studies show, therefore, that there is an increase in this ratio because of a slightly greater effect on synthesis which is not reflected in secretion. Similarly, hydrocortisone exerts a greater increase in transferrin synthesis than secretion and causes the ratio of synthesis to secretion to increase from 3.6 to 6.2. We propose that during continued subculturing a Hepa-2 variant is selected in which the regulation of serum glycoprotein synthesis and secretion is uncoupled. Furthermore, this effect is specific for secretory glycoproteins since the regulation of albumin synthesis and secretion by hydrocortisone and cyclic nucleotides remained unaltered.

Benzo[a]pyrene diol epoxide DNA adduct formation in transformable and non-transformable human foreskin fibroblast cells in vitro.

The uptake of benzo[a]pyrene (BP) by low passage (LP) and high passage (HP) human skin fibroblast cells is followed by its transport into the nucleus as the parent compound. When the LP and HP cells were treated with BP for 24 h and the DNA was isolated and enzymatically digested, several DNA adducts were detected. In both the LP and HP cells a small amount of the radiolabel was associated with the 7 beta-BPDE-I-dG, 7 alpha-BPDE-I-dG and BPDE-II-dG adducts. Although there were no major qualitative differences in the adducts formed in the LP and HP cells, a higher proportion of the radiolabel was associated with the 7 beta-BPDE-I-dG adduct in the LP cells. When LP or HP cells were treated with BPDE-I, the ultimate carcinogenic form of BP, similar levels of DNA modification were observed in the two cell types and the h.p.l.c. profiles of these adducts were essentially identical. BPDE-I induced a carcinogenic event in the LP but not the HP cells as measured by anchorage independent growth in soft agar and cellular invasiveness of the chick embryonic skin organ cultures.

Inhibition of sindbis virus replication by ribavirin: Influence of cultural conditions and of the host cell phenotype

Several factors which influence the ability of ribavirin to inhibit the replication of Sindbis virus in baby hamster kidney (BHK) cells are described; these include the cell passage level, the presence of serum in the medium and cell density. Inhibition of viral replication was much more effective with low passage cells than with high passage cells. However, with high passage cells antiviral activity was enhanced 1) when subconfluent cultures were infected, rather than confluent cultures, and 2) when infected cultures were maintained in the presence of serum rather than without serum. Under all conditions, a good correlation was found between the inhibition of virus replication by ribavirin and its ability to reduce cellular GTP levels. Using Chinese hamster ovary cell mutants we found that ribavirin inhibited Sindbis virus replication in cells lacking hypoxanthine phosphoribosyltransferase (HPRT −) but not in HPRT + cells. This result and the demonstration that Aedes albopictus cells (in which ribavirin is very effective) are unable to incorporate hypoxanthine suggest that certain salvage enzymes involved in purine metabolism can interfere with the antiviral activity of ribavirin.

Basal Lamina Formation by Normal and Transformed Mouse Mammary Epithelial Cells Duplicated in Vitro<xref ref-type="fn" rid="fn2">2</xref><xref ref-type="fn" rid="fn3">3</xref>

Cells from low-passage (LP) cultures of a mouse mammary epithelial line (NMuMG cells) form a basal lamina when they are cultured on a type I collagen gel substratum. A high-passage (HP) strain of this line maintained the morphologic, serologic, and karyologic properties of the LP cells. For the determination of whether transformation of the NMuMG cells might lead to defects in the basal lamina, cells from LP cultures were compared in vivo and in vitro with cells of HP cultures for tumorigenicity, growth characteristics, and ability to form a lamina. The LP NMuMG cells had a typical epithelial morphology and showed no cytologic evidence of cancer. They formed an ultrastructurally normal continuous basal lamina in vivo when they were injected into athymic nude mice. In contrast, the HP cells were pleomorphic and highly invasive when injected into nude mice where they showed frequent and large basal lamina defects. These cells also accumulated only traces of lamina-like materials when cultured on a collagen gel, indicating that neoplastic transformation had markedly reduced the ability of NMuMG cells to form a basal lamina both in vivo and in vitro. Because the collagen gel culture system duplicated the in vivo situation with regard to basal lamina integrity, the basis for this lack of in vitro basal lamina formation may be physiologically relevant for the mechanism of malignant invasion.

Enhancement of melanotic expression in cultured mouse melanoma cells by retinoids.

Retinoic acid (RA), which reduces the rate of cell proliferation in S91 mouse melanoma clone C2 cells, was found to stimulate the expression of their melanotic phenotype. RA treatment also induced the extension of long cellular processes. The RA effects on melanogenesis included stimulation of tyrosinase activity and augmentation of cellular melanin content to levels 3- to 4-fold higher than in untreated cultures at similar cell densities. These effects became apparent after 48 hours of exposure to 10(-5) M RA and increased thereafter. Half-maximal stimulation in cells treated for 6 days occurred at 5 X 10(-7) M RA. Although the degrees of melanogenesis enhancement by RA (10(-5) M) and by alpha-melanocyte stimulatory hormone (2 X 10(-7) M) were similar, the former did not alter the intracellular cAMP level, whereas the latter induced a transient 4-fold increase. In high-passage (p28) cells, as well as in low-passage cells (less than p10) treated with tyrosinase inhibitor phenylthiocarbamate, melanin synthesis was suppressed in the absence and presence of RA, yet the ability of RA to inhibit cell proliferation was not compromised. In the presence of the tumor promotor phorbol myristate acetate (greater than 5 X 10(-9) M) melanin synthesis in control as well as in cells exposed to RA was dramatically inhibited. Phorbol which is not active in tumor promotion had no effect on melanogenesis. In addition to RA, other retinoids, such as 13-cis-retinoic acid, retinyl acetate, the TMMP analog of RA and the phenyl analog of RA, but not the pyridyl analog of RA or retinyl palmitate, also inhibited cell growth and enhanced melanin synthesis.

Analysis of intracellular distribution and binding of benzo[ a]pyrene in human diploid fibroblasts

Previous work with low passage synchronized human foreskin fibroblast cell populations has indicated that benzo[ a]pyrene (BP) can induce a carcinogenic event [3]. BP additionally has shown to damage DNA in logarithmically growing low passage cultures [9]. High passage cells, on the other hand, seem to be refractory to transformation by BP, even though this agent can induce DNA damage, similar to that seen in low passage cells. When low passage cells were treated with BP, the initial binding of the hydrocarbon was primarily to a cytoplasmic protein complex of molecular weight 12,500, while in high passage cells, a major portion of BP was bound to a protein complex of molecular weight 200,000. High-pressure liquid chromatography (HPLC) profiles of ethyl acetate extractable fractions from the BP-cytoplasmic protein complexes of low and high passage cells demonstrated that the majority of the BP remained unmetabolized. When nuclei were isolated from low and high passage cells prior to the HPLC analysis, the major component (90%) was again unmetabolized BP. The results suggest selective attachment of BP to different cytoplasmic protein complexes of logarithmically growing human diploid fibroblast cells dependent on the passage level of the cells.

Properties of RBL-5 leukemia cells cultivated in vitro.

RBL-5 leukemia induced by Rauscher murine leukemia virus (R-MuLV) carried in the ascites form in C57BL/6 mice was cultivated in vitro and passaged continuously. Both low- and high-passage cells released the same level of murine leukemia virus, as detected by the XC plaque assay. Membrane antigens of the cultivated cells were also detected by the indirect immunofluorescence assay. Both low- and high-passage cells showed the same activity of antigen detectable by anti-R-MuLV serum, whereas only the high-passage cells reacted strongly against a syngeneic antiserum to RBL-5 crude membrane (CM). The oncogenicity of the low and high passages of the cultivated cells was compared with the ascites cells. The results indicated that the oncogenic potential of the cells gradually decreased during in vitro passage. The accumulation or retention of CM antigen on the membrane of the high-passage cells suggested that their decreased oncogenic potential might be due to increased immunogenicity and/or immunosensitivity. This strengthened the hypothesis that the cellular transplantation antigens are not identical to type C viral antigens, since the R-MuLV antigens were not altered during passage in vitro.

Source, alterations, characteristics and use of a new dog cell line (Cf2Th)

A cell line derived from normal fetal canine thymus (Cf2Th) has been in culture since 1967. During cultivation the cells have changed morphologically from a fibroblast-like to flat, fusiform appearance and karyologically from diploid (2n=78) with 76 telocentric autosomes to hypodiploid with newly formed atelocentric chromosomes. The cells retain canine characteristic enzyme activity (G6PD and LDH) as well as cell membrane fluorescence and are free of mycoplasma. High passage cells produce tumors in ATST mice. No endogenous viruses have been detected in these cells. No original publication exists, to date, on the origin of this line, but seed stocks thereof have been distributed to many laboratories and the cells have served as experimental substrates in a number of published works on oncology albeit under different designations. The present information is offered in order to establish the provenance of this valuable cell line and to list characteristics which may serve to monitor for its purity and to distinguish it from other existing cell lines of dog origin also in common use.

The cell cycle of low passage and high passage human diploid fibroblasts

Using mitotic selection, synchronous populations of passage 25 and passage 50 WI38 cells were obtained. Thymidine incorporation, mitotic index, and labeling index were determined. No great differences could be seen in the organization of the cell cycle of low passage and high passage cells. Although labeling indices for passage 25 and passage 50 exponential cells were 83% and 37%, respectively, 75% of all mitotically selected cells were found to enter S phase, regardless of passage number. Thus, the loss of late passage diploid cell cultures does not occur from alteration of the cell cycle in the majority of rapidly proliferating cells.