通过高表达Igf1/Bcl-2或Bcl-2/Cyclin E基因组合使CHO细胞适于在无蛋白培养基中抗凋亡培

哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中,哺乳动物细胞生长活力差,且容易发生细胞凋亡,因而难以大规模培养。为解决此问题,应用双顺反子表达载体在CHO-dhfr-细胞中同时表达Igf-1/Bcl-2或Bcl-2/Cyclin E基因组合,通过Bcl-2使细胞获得抗凋亡能力;通过Igf-1或Cyclin E促进细胞生长分裂,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO-dhfr^-细胞,应用Western blot从G418抗性克隆中分别筛选到Bcl-2高表达克隆若干个,对其中表达Bcl-2最高的CHO-IB3和CHO-BC1做进一步Western blot和流式细胞分析,确认此两个细胞株分别高表达Igf-1/Bcl-2和Bcl^-2/Cyclin E基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡,并以流式细胞术和DNA Ladder法检测细胞凋亡,证明CHO-IB3和CHO-BC1均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO-dhfr-对照细胞。在细胞培养瓶中的连续培养实验表明,CHO-IB3和CHO-BC1在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHOdhfr-对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长,适于作为生物工程宿主细胞。     

通过高表达Igf1/Bcl-2或Bcl-2/Cyclin E基因组合使CHO细胞适于在无蛋白培养基中抗凋亡培养

哺乳动物细胞表达系统是生产重组蛋白药物最常用的表达系统。但在无蛋白培养基中,哺乳动物细胞生长活力差,且容易发生细胞凋亡,因而难以大规模培养。为解决此问题,应用双顺反子表达载体在CHO-dhfr^-细胞中同时表达Igf-1／Bcl-2或Bcl-2／CyclinE基因组合,通过Bcl-2使细胞获得抗凋亡能力;通过1gf-1或CyclinE促进细胞生长分裂,使细胞获得在无蛋白培养基中生长的能力。以上述基因组合转染CHO-dhfr^-细胞,应用Western blot从G418抗性克隆中分别筛选到Bcl-2高表达克隆若干个,对其中表达Bcl-2最高的CHO-IB3和CHO-Bcl做进一步Western blot和流式细胞分析,确认此两个细胞株分别高表达Igf-1／Bcl-2和Bcl-2／CyclinE基因组合。分别通过撤去血清和加入放线菌素D诱导细胞凋亡,并以流式细胞术和DNA Ladder法检测细胞凋亡,证明CHO-IB3和CHO一BCl均具有较强的抗细胞凋亡能力。MTT法证明两个细胞株在不含血清的IMDM培养基中的增殖活力显著高于CHO-dhfr^-对照细胞。在细胞培养瓶中的连续培养实验表明,CHO-IB3和CHO-BCl在本实验室设计的IMEM无蛋白培养基中的生长速度和活细胞数显著高于CHO-dhfr^-对照细胞。提示此两个细胞系能够在无血清培养基中抗凋亡高活力生长,适于作为生物工程宿主细胞。     

细胞周期蛋白D1特异性核酶表达载体的构建及其活性

应用mfold程序对锤头状核酶(ribozyme,Rz)和大鼠细胞周期蛋白(cyclin)D1基因的二级结构进行分析,设计合成锤头状Rz基因,通过RT-PCR扩增获得大鼠细胞周期蛋白D1目的基因,将Rz基因和细胞周期蛋白D1基因分别克隆入载体pGEM-3Zf( )中,体外转录Rz基因和靶基因并进行切割实验;将Rz基因与逆转录病毒载体pLXSN重组得到Rz真核表达载体pLXSN-Rz,将其转染入HSC-T6细胞,G418筛选出阳性细胞克隆,用RT-PCR检测细胞周期蛋白D1基因的表达。结果显示:针对目的基因的832位点设计合成了Rz832,成功获得Rz832基因、细胞周期蛋白D1mRNA的体外转录载体pGEM3Zf-Rz832和pGEM3Zf-cD1,经体外转录出Rz832(105nt)及细胞周期蛋白D1mRNA(1079nt)。体外切割实验证实Rz832能够特异性切割细胞周期蛋白D1mRNA,产生1014nt和65nt的切割产物,切割效率为80%。所构建的pLXSN-Rz832经酶切电泳、PCR鉴定显示,插入的Rz832序列大小约为57bp,与预期结果相同,经测序证实Rz832序列正确。转染pLXSN-Rz832的肝星状细胞(hepaticstellatecells,HSCs)细胞周期蛋白D1mRNA的表达受到明显抑制,仅为对照组的42.22%(t=-193.443,P<0.01),结果表明:Rz832能够在体外特异性切割细胞周期蛋白D1mRNA、并在HSC-T6细胞内有效抑制细胞周期蛋白D1基因的表达。     

KAI1基因转染人乳腺癌细胞系的建立及初步研究

目的:通过将外源性KAI1基因转染入高转移性人乳腺癌细胞株,为乳腺癌基因治疗的实验室研究提供靶细胞,并初步探讨该基因对乳腺癌细胞增殖能力及细胞周期的影响。方法:采用脂质体法将pCMV-KAI1质粒转染入低表达KAIl基因的人乳腺癌细胞株MDA-MB-231中,经G418筛选后获得抗性克隆,利用RT-PCR、Western blot分析目的基因及其蛋白的表达情况,并利用MTT法和平板克隆形成实验初步探讨该基因对乳腺癌细胞体外增殖能力的影响,流式细胞术检测细胞生长周期的变化。结果:稳定转染KAI1基因的细胞株中有外源性目的基因和相应蛋白的高表达;MTT法示细胞增殖力下降,转染KAI1基因的集落形成率(25.33 2.36)％较转染前(43．17 2．75)％明显降低(P<0．05),流式细胞术显示转染KAI1基因后G1／G0期细胞数量由未转染前的(36．78 0．61)％升高至(64 7．56)％,M／G2期细胞数量则由(17．88 0．76)％降至(7．63 0．60)％,差异有显著性。结论:通过脂质体转染法获得了高表达KAI1基因及其蛋白的人乳腺癌细胞株,并发现该细胞株的体外增殖能力明显下降,这可能是KAI1基因通过调节细胞周期来实现的。     

在CHO细胞中表达重组sPDGFRα-Fc及其抑制细胞增殖的研究

目的：筛选在CHO-K1中高表达sPDGFRα-Fc的重组细胞株,并对分泌到培养基的表达产物进行抑制细胞增殖的活性分析。方法：构建带有Fc标签的sPDGFRα基因重组表达载体 pIRES-Neo3-sPDGFRα-Fc;在脂质体介导下,转染CHO-K1细胞,G418筛选2周后获得若干单克隆细胞株,随机挑取单克隆细胞进一步放大培养,RT-PCR筛选阳性单克隆细胞;Real-Time PCR方法鉴定各阳性细胞株中的sPDGFRα-Fc基因的转录水平, Western blot检测进一步验证各细胞中目的蛋白表达水平;筛选出表达最高的细胞株,更换无血清培养基培养,取含有可溶性sPDGFRα的培养基上清冻干浓缩,MTT法检测目的蛋白的抑制细胞增殖能力。结果：成功构建重组表达载体并在CHO-K1中成功表达,各阳性单克隆细胞株的表达量有差异且在转录水平和蛋白表达水平表现一致,从无血清培养基中收集的可溶性sPDGFRα-Fc明显抑制血管内皮细胞的增殖。结论：成功筛选获得CHO-K1中高表达sPDGFRα-Fc的重组细胞株,获得的可溶性sPDGFRα-Fc能抑制细胞增殖,有望成为治疗因PDGF及其受体引起的多种疾病的药物。     

siRNA-cyclin D1对乳腺癌MCF-7细胞增殖的抑制作用

研究小干扰RNA(small interfering RNA,siRNA)对乳腺癌MCF-7细胞株cyclin D1表达的抑制及对细胞增殖的影响。化学合成针对cyclin D1基因的siRNA,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin D1 mRNA和蛋白的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。在实验中,10、50、100 nmol/L siRNA-cyclin D1分别使MCF-7细胞cyclin D1 mRNA表达降低了57．85%、63．22%和68．02%,蛋白表达降低了51．13%、62．09%、77．68%。转染siRNA-cyclin D1后,细胞增殖受到抑制,细胞周期阻滞于G1期,软琼脂克隆形成率降低。结果提示siRNA可以有效抑制MCF-7细胞株中cyclin D1的表达,使细胞周期阻滞于G1期,从而抑制细胞增殖。     

小鼠DLL1真核表达载体的构建及其在肿瘤细胞中的表达

目的:构建带绿色荧光蛋白的小鼠DLL1全长基因真核表达载体,并在肿瘤细胞中表达。方法:利用PCR特异性引物扩增出DLL1基因全长,将克隆的基因片段插入带绿色荧光蛋白的真核表达载体pIRES2-EGFP质粒中。然后利用脂质体将重组质粒pIRES2-EGFP-DLL1转染进小鼠B16黑色素瘤细胞中,并通过G418筛选后选取生长良好、荧光强度高的三株单克隆进行mRNA水平DLL1表达的鉴定。结果:成功扩增小鼠DLL1的全长基因。克隆入质粒载体后,通过DNA序列测定证实其序列正确。将构建的pIRES2-EGFP-DLL1质粒转染小鼠B16黑色素瘤细胞,经过G418筛选和荧光显微镜观察后,挑选得到GFP阳性率90%以上的稳定转染细胞株。RT-PCR检测稳定转染细胞的mDLL1的表达显著增加,进一步证实了pIRES2-EGFP-DLL1的表达效能。结论:成功构建了小鼠DLL1基因的真核表达质粒,证实其在真核细胞B16中可以表达。     

HSP90高表达细胞株的建立及细胞增殖性状的改变

目的:建立稳定高表达热休克蛋白90(HSP90)细胞株,研究其对细胞增殖的影响.方法:含人HSP90 13全长基因的重组质粒pSmycHSP经亚克隆、纯化、酶切鉴定后,用电穿孔法转染到小鼠成纤维细胞系NIH-3T3细胞内.经G418筛选、克隆分离培养,用免疫细胞化学、免疫印迹鉴定阳性克隆.以转染空质粒的NIH-3T3细胞为对照,用MTT法、流式细胞术测定,分析HSP90高表达对细胞增殖和细胞周期的影响.结果:转染pSmycHSP的NIH-3T3细胞HSP90染色增强,生长速度减慢,S期DNA含量降低.结论:己建立稳定高表达热休克蛋白90(HSP90)NIH-3T3细胞株;转染pSmycHSP的NIH-3T3细胞能够有效地表达HSP90,影响细胞周期,使细胞增殖迟滞.     

CREG基因敲除小鼠胚胎干细胞的筛选及鉴定

目的:为阐明E1A激活基因阻遏子(Cellular repressor of E1A-stimulated genes,CREG)在发育过程中的作用,本研究拟通过高浓度药物筛选获得基因敲除的小鼠胚胎干细胞(Embryonic stem cell,ESC)。方法:用0.5 mg/ml、1.0 mg/ml、1.5 mg/ml、2.0mg/ml、2.5 mg/ml及3.0 mg/ml 6个浓度的G418培养CREG杂合型(CREG其中一个等位基因被新霉素抗性neo基因替代)小鼠ESC 2周,确定最佳的G418筛选浓度。挑取该浓度下存活的ESC克隆进行扩增。将每个ESC克隆一半冻存,另一半贴壁培养。待ESC生长至80%融合后分别提取基因组DNA和蛋白。PCR方法扩增CREG基因明确基因组中是否存在CREG基因,WesternBlot方法鉴定是否有CREG蛋白表达。结果:确定2.0 mg/ml G418为最佳的筛选浓度。在该浓度下,共获得存活的克隆10个,PCR证实C2及C7克隆基因组中没有CREG基因,Western Blot证实C2及C7无CREG蛋白表达。结论:成功获得CREG基因敲除的小鼠ESC 2株,为深入研究CREG功能奠定了基础。     

LTF基因在鼻咽癌细胞系中生物学功能的初步研究

目的:初步探讨野生型LTF基因在鼻咽癌细胞系中的生物学功能。方法:野生型LTF导入鼻咽癌细胞系,G418筛选,RT-PCR和Western-blotting分别在mRNA和蛋白质水平进行验证,得到稳定表达LTF基因的鼻咽癌细胞系。流式细胞术、平板克隆形成实验和MTT法分别检测细胞周期、细胞的克隆形成能力和细胞生长曲线。结果:成功导入LTF并稳定表达的鼻咽癌细胞系,G0-G1期细胞百分比例明显增加(72.01%vs 62.31%),G2-M期细胞百分比例减少(6.26%vs 10.81%);克隆形成能力降低(39.5%vs 59.7%),体外瘤细胞增殖能力降低(P0.05)。结论:LTF基因可阻滞细胞周期、抑制鼻咽癌细胞系的增殖能力和克隆形成率,同时为进一步的体内试验研究奠定基础。     

Maintenance of growth transformation with Epstein-Barr virus is mediated by secretion of autocrine growth factors in two serum-free B-cell lines.

The characteristics of two tamarin (Saguinus oedipus) B-cell lines (sfBIT and sfBT) growth-transformed by Epstein-Barr virus (EBV) that proliferate continuously in serum-free medium are described. sfBIT was established by selecting cells for growth in RPMI 1640 supplemented with insulin, transferrin, and selenium (J. E. Shaw, R. G. Petit, and K. Leung, J. Virol. 61:4033-4037, 1987). sfBT, a subline of sfBIT cells reported here for the first time, required transferrin as the only protein supplement for continuous growth in RPMI 1640. Growth of sfBT cells was linear with human transferrin at 10(-2) to 10 micrograms/ml. Transferrin at 5 micrograms/ml yielded a culture density of 5 X 10(5) to 1 X 10(6) cells per ml, a cell doubling time of 2 to 3 days, and a culture viability greater than 95%. sfBIT and sfBT cells released transforming virus during continuous growth in serum-free culture medium without EBV-inducing agents. The spent medium of both serum-free lines supported cell growth at low culture density (1 x 10(4) to 5 X 10(4) cells per ml), but growth was arrested at low culture density with fresh serum-free medium. A procedure to measure growth-promoting activity (GPA) was established, and it revealed that the GPA of spent medium was greater than that of fresh medium for both serum-free cell lines. When fresh and spent media were dialyzed (molecular weight cutoff, 3,500) and subsequently concentrated by lyophilization, only the GPA of spent medium increased. We conclude that maintenance of growth transformation of tamarin cells latently infected with EBV is mediated by growth factors that are entirely autocrine in origin.     

Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity,cell survival,and mitochondria number in CHO-DG44 grown in suspension and serum-free media

We have engineered dihydrofolate reductase-negative (dhfr-/-) Chinese hamster ovary (CHO) DG44 cells adapted for growth in serum-free suspension cultures for simultaneous expression of the common cold therapeutic, the soluble intercellular adhesion molecule 1 (sICAM), and the antiapoptosis determinants bcl-2 or bcl-x(L). Detailed analyses of titer and antiapoptosis characteristics of these production cell lines included an independent (sICAM; bcl-2/bcl-x(L)) as well as a cocistronic (sICAM-(bcl-2/bcl-x(L))) expression set-up in which translation-initiation of the survival cistron is driven by an internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). In transient transfections or stable mixed populations and in comparison to isogenic sICAM-only control vectors, both bcl-x(L)-encoding configurations achieved higher sICAM yields while bcl-2 over-expression resulted in decreased product levels. Overall, the death-protective impact of bcl-2 and bcl-x(L) in engineered CHO-DG44 was not significant under typical batch-mode operation, an observation that was confirmed by clonal analysis. bcl-2 and bcl-x(L) displayed their antiapoptosis potential only following dhfr-based amplification in sICAM-producing CHO-DG44 cell lines. In all cases, bcl-x(L) outperformed bcl-2 in its cell death-protective capacity. Amplification-dependent high-level expression of mitochondria-localized bcl-2 family members required for successful antiapoptosis engineering may be essential to compensate for increased mitochondria numbers found to be associated with production cell lines grown in serum-free medium.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Coexpression of acidic fibroblast growth factor enhances specific productivity and antibody titers in transiently transfected HEK293 cells

Recombinant proteins are of great commercial and scientific interest. However, most current production methods using mammalian cells involve the time- and labor-intensive step of creating stable cell lines. Although production methods based on transient gene expression could offer a significant improvement, transient transfection is currently still limited by low titers and low specific productivity compared to stable cell lines. To overcome these bottlenecks, we have explored the use of various growth factors to enhance specific productivity and titers in the context of transient gene expression. For that purpose, several growth factors were cloned and screened for their effect on transient gene expression in HEK293E and CHO-DG44 cells. In particular, acidic fibroblast growth factor (aFGF) was able to increase specific productivity by 60% and recombinant protein titers by 80% in HEK293E cells, while FGF9 increased titers by 250% in CHO-DG44 cells.     

The growth requirements of BHK-21 cells in serum-free culture

A serum-free medium for serial culture of baby hamster kidney cell line 21 (BHK-21) as container-adherent cells was developed. The medium is a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium supplemented with fibroblast growth factor, fibronectin, insulin, oleic acid (preincubated with fatty-acid-free bovine serum albumin as carrier), and transferrin. The fibronectin was required for cell adherence, the other factors for optimal cell multiplication. When cell input was greater than about 1,900 cells/cm², this serum-free medium supported cell multiplication at a rate approximately equal to the rate in medium with 10% serum. At lower cell input, growth in the serum-free medium was poor unless it was supplemented with serum-free medium which had been conditioned by BHK-21 cells. The conditioned medium contained a factor(s) which enabled or stimulated cell multiplication.     

Growth of human malignant lymphoid cell lines in serum-free medium

Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products.     

Correlation of the inability to sustain growth in defined serum-free medium with the suppression of tumorigenicity in Wilms' nephroblastoma.

We report the investigation of the growth properties of tumorigenic and reverted nontumorigenic Wilms' nephroblastoma cells when cultured in serum-free medium. Wilms' tumor, a pediatric nephroblastoma, has been associated with deletions encompassing the p13 band of chromosome 11 and an independent loss of heterozygosity at 11p15. Weissman et al. (Science 236:175-180, 1987) transferred a human der(11) chromosome into the G401.6TG.6 Wilms' tumor cell line via the microcell-mediated chromosome transfer technique. The resulting microcell hybrids were nontumorigenic when assayed in nude mice; however these cells retained all of the in vitro growth and morphological characteristics of the tumorigenic parental cells in 10% fetal calf serum (FCS). Segregation of the der(11) chromosome from the nontumorigenic microcell hybrid cells resulted in the reappearance of the tumorigenic phenotype in vivo. In vitro culture of these cell lines in serum-free medium supplemented with 0.1% bovine serum albumin (BSA) and 10 ng/ml Na2O3Se resulted in sustained growth of both the tumorigenic parent and the tumorigenic segregant while the nontumorigenic microcell hybrids were unable to divide. The separate addition of either 10 ng/ml of epidermal growth factor (EGF) or 5 micrograms/ml of insulin did not alter this effect. However, the addition of 5 micrograms/ml of transferrin stimulated the nontumorigenic microcell hybrid cells to grow at a rate comparable to the tumorigenic cells. In addition, conditioned serum-free medium from the tumorigenic parental or tumorigenic segregant cell lines was able to stimulate the growth of the nontumorigenic microcell hybrid cells, whereas the reciprocal experiment had no effect on the growth of the tumorigenic cells. These data suggest that the inability of the microcell hybrid cells to grow in serum-free conditions is correlated with their genetic nontumorigenic phenotype and that a specific growth factor, transferrin, can bypass or alter this negative growth regulatory pathway(s) in vitro.     

Characterization of a serum-free culture system comparing growth factor requirements of transformed and untransformed cells

We describe the first completely serum-free model culture system for comparing growth control in transformed and untransformed cells. Continuous maintenance of untransformed AKR-2B fibroblasts and chemically transformed AKR-MCA cells in the presence of serum-free medium containing epidermal growth factor (E), insulin (I), and transferrin (T) resulted in cell lines which proliferated with similar doubling times (14 h), comparable to parental lines maintained in 10% serum (16 h). The transformed MCA-SF cells and untransformed AKR-SF cells did not differ in their saturation densities in medium containing E + I + T. However, the monolayer proliferation of MCA-SF cells was significantly greater than that of the AKR-SF cells in the presence of E + T, I + T, or T alone. Both cell lines required T to proliferate in monolayer culture. [3H]-Thymidine incorporation experiments and autoradiographic analysis indicated that quiescent MCA-SF cells could reenter the cell cycle by addition of nutrients alone. The combination of E + I + T produced no additional stimulation of DNA synthesis. In contrast, individual polypeptide growth factors (E, I, IGF-I, PDGF, FGF a or b, or TGF-beta 1) were required to elicit a mitogenic response in the untransformed AKR-SF cells. Peak mitogenesis occurred from 18-20 h for all growth factors except TGF-beta 1 (32 h). Neither AKR-SF nor MCA-SF cells could grow with anchorage independence in serum-free medium, unless both TGF-beta 1 and FGF a or b were simultaneously present. The results indicate that this well-defined, serum-free model system can be utilized to detect growth factor-related alterations associated with the transformed state.     

Serum inhibition of proliferation of serum-free mouse embryo cells

Serum-free mouse embryo (SFME) cells, derived in medium supplemented with insulin, transferrin, high density lipoprotein, epidermal growth factor, and fibronectin, do not undergo crisis, maintain a predominantly diploid karyotype with no detectable chromosomal abnormalities for well over 100 population doublings in vitro, and are growth inhibited by concentrations of serum that are growth-stimulatory for most cell lines in culture. Serum inhibition of SFME cell proliferation was reversible and was not prevented by addition of the supplements of the serum-free medium, even when added repeatedly during the culture period. The serum effect on SFME cell proliferation could be detected after incubation in serum-containing medium for as little as 8 h. SFME cells in serum-containing medium were arrested in the G1 phase of the cell cycle with a greatly reduced rate of incorporation of precursors into DNA and thymidine kinase activity, while a reduction in rate of incorporation of amino acids into protein was not observed. SFME cultures maintained for extended periods in serum-containing medium underwent a crisis-like period followed by the appearance of variant cells capable of growing in serum-supplemented medium. These cells exhibited abnormal karyotype and were resistant to several inhibitors of proliferation active on the parent SFME cell type.