Chinese Hamster Ovary Sublines Research Articles

Abstract 3980: Dual targeting mitochondrial and cytosolic one-carbon metabolism via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates

Abstract Cellular one-carbon (1C) metabolism generates a host of metabolites especially critical to cancer cells. Classical 1C inhibitors often primarily target a single enzyme, commonly leading to drug resistance and necessitating the development of novel compounds targeting multiple enzymes. We synthesized several novel 5-substituted pyrrolo[3,2-d]pyrimidine compounds (AGF291, AGF320, and AGF347) with transport by the proton-coupled folate transporter (PCFT), a folate transporter with a narrow physiological niche, but commonly expressed in many solid tumors. These analogs showed substantial inhibition in proliferation assays with Chinese hamster ovary sublines engineered to individually express PCFT or the ubiquitously-expressed reduced folate carrier (the major tissue folate transporter), as well as in the PCFT-expressing human cancer cell lines H460 (large cell lung carcinoma), HCT-116 (colorectal carcinoma), and MIA PaCa-2 (pancreatic ductal adenocarcinoma). Full abrogation of inhibitory effects for all compounds required co-treatment with both adenosine and glycine, suggesting dual-targeting of cytosolic de novo purine biosynthesis and mitochondrial 1C/glycine metabolism. For cytosolic de novo purine biosynthesis, lack of protection by 5-aminoimidazole-4-carboxamide (AICA) suggested AICA ribonucleotide formyltransferase (AICARFTase), the second folate-dependent enzyme, as the likely enzyme target. AICARFTase inhibition was confirmed in intact H460 and HCT-116 cells by measuring accumulation of AICAR by targeted metabolomics. Targeting of mitochondrial enzymes in H460 and HCT-116 cells was suggested by selective inhibition of incorporation of [3-14C]serine over [14C]formate into the purine intermediate [14C]formyl glycinamide ribonucleotide. [2,3,3-2H]Serine isotope label scrambling analysis confirmed the mitochondrial target to be serine hydroxymethyltransferase2 (SHMT2). SHMT2 is a potentially promising anticancer drug target as its expression is highly correlated with the malignant phenotype across a broad spectrum of cancers beyond lung and colon, including breast, glioma, and liver. Indeed, our initial in-vivo studies with AGF291 suggest potent efficacy toward both MIA PaCa-2 and H460 xenograft mouse models. Our studies demonstrate the potential for tumor-selective dual targeting of both mitochondrial and cytosolic 1C metabolism to overcome resistance to classical 1C inhibitors. Citation Format: Aamod S. Dekhne, Gregory S. Ducker, Josephine Frühauf, Khushbu Shah, Md. Junayed Nayeen, Adrianne Wallace-Povirk, Carrie O'Connor, Zhanjun Hou, Lisa Polin, Aleem Gangjee, Joshua D. Rabinowitz, Larry H. Matherly. Dual targeting mitochondrial and cytosolic one-carbon metabolism via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3980.

Fusion of the Dhfr/Mtx and IR/MAR Gene Amplification Methods Produces a Rapid and Efficient Method for Stable Recombinant Protein Production

Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.

Abstract 3824: Design, synthesis and biological evaluation of 4-atom side chain pemetrexed (PMX) homolog AG127 as a potent inhibitor of tumors expressing folate receptors via 5-aminoimidazole-4-carboxamide ribonucleotide formyl transferase (AICARFTase) inhibition

Abstract Two major hurdles in cancer therapy are the inability of chemotherapeutic agents to selectively target tumor cells over normal tissues, and the development of tumor cell resistance. The multi-enzyme-targeted anticancer drug pemetrexed (PMX) suffers from dose-limiting toxicity, in part due to its membrane transport by the reduced folate carrier (RFC) which is ubiquitously expressed in normal and tumor cells. We recently described a novel series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates. A highly active compound of this series (AG71) included a 4-carbon bridge and was selectively transported into cells by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) but not RFC. AG71 inhibited de novo purine biosynthesis at the level of glycinamide ribonucleotide formyltransferase (GARFTase). AG71 potentially inhibited proliferation of FR- and PCFT-expressing Chinese hamster ovary cells and of both KB and IGROV1 tumors in vitro; in SCID mice with subcutaneous KB tumors, AG71 was highly active against both early and advanced stage tumors. Based on the 5-substituted pyrrolo[2,3-d]pyrimidine scaffold of PMX and our results with the 6-substituted compound AG71, we synthesized 5-substituted pyrrolo[2,3-d]pyrimidine phenyl analogs with 1- to 6-carbon atoms in the bridge (AG124-AG129). Analogs were tested for anti-proliferative effects on KB human tumor cells and on Chinese hamster ovary (CHO) sublines engineered to individually express RFC (PC43-10), PCFT (R2/PCFT4), and FRα (RT-16). The 4-carbon atom chain analog (AG127) showed IC50 value of 1.9 nM, only slightly less than for AG71 (0.55 nM), in inhibiting proliferation of KB tumor cells in culture. In the CHO sublines, both AG127 and AG71 inhibited growth of RT-16 cells (IC50 = 3.1 nM and 1.8 nM respectively). AG71 was inactive toward PC43-10 cells and was inhibitory toward R2/PCFT4 cells (IC50 = 43.4 nM). Compared to RT-16 cells, AG127 showed reduced inhibitory activity toward PC43-10 cells (IC50 = 54 nM) and R2/PCFT4 cells (IC50 = 733 nM). PMX is a multi-targeted agent with its principle inhibitory effects on thymidylate synthase and secondary inhibitory effects on AICARFTase and GARFTase in de novo purine nucleotide biosynthetic. Inhibition of KB cell proliferation by AG127 was abolished by excess adenosine, but not thymidine or AICA, suggesting that AICARFTase was the principle intracellular target. Collectively, our results show that the side chain homologated analog of PMX, AG127, exhibits nanomolar inhibition of proliferation of KB tumor cells in culture. The unique and specific AICARFTase inhibition of AG127 makes it a promising option for cancer chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3824. doi:1538-7445.AM2012-3824

Abstract 3822: A tumor-targeted 5-pyrrolo[2,3-d]pyrimidine antifolate is a selective substrate for folate receptor ≤ and potent inhibitor of 5-amino-4-carboxamide formyltransferase in de novo purine nucleotide biosynthesis

Abstract Clinically used antifolates are transported into cells by three major systems including the reduced folate carrier (RFC), folate receptor (FR)α, and proton-coupled folate transporter (PCFT). RFC is ubiquitiously expressed in normal tissues, however, expression of FRα and PCFT is more limited. FRα and PCFT are expressed in human tumors, suggesting their potentials for tumor targeting with cytotoxic antifolates selective for these systems. We previously identified 6-substituted pyrrolo[2,3-d]pyrimidine benozyl antifolates characterized by 3 (AG17) and 4 (AG23) carbons in the bridge region with potent antiproliferative activities toward human tumor cells. AG17 and AG23 were selectively transported by FRα over RFC and inhibited de novo purine biosynthesis at α-glycinamide ribonucleotide formyltransferase (GARFTase). We synthesized the corresponding 5-substituted pyrrolo[2,3-d]pyrimidine benzoyl antifolates with 2 to 6 bridge carbons and tested growth inhibition of Chinese hamster ovary sublines that express human FRα (RT16), PCFT (R2/PCFT4), or RFC (PC43-10). Studies were extended to include KB human tumor cells expressing all three transporters. The 5-substituted analog (AG127) with a 4 carbon bridge was the most active of the series and inhibited proliferation of FRα-expressing RT16 (IC50 = 3.1 nM) and KB (IC50 = 1.9 nM) cells. Inhibitory effects of AG127 toward RFC-expressing PC43-10 and PCFT-expressing R2/PCFT4 cells were substantially reduced (IC50s of 54 and 733 nM, respectively), establishing FRα selectivity. In KB cells, AG17, AG23 and AG127 bound to FRα with high affinity. The antiproliferative effects of both 5- and 6-substituted analogs (AG127 and AG17/AG23, respectively) were protected by excess adenosine but not thymidine at drug concentrations up to 1 μM. Whereas 5-amino-4-imidazole carboxamide (AICA) completely protected KB cells from the effects of AG17/AG23, establishing GARFTase inhibition, AICA protection was incomplete for AG127. This suggests that AG127 inhibits AICA ribonucleotide formyltransferase (AICARFTase), the 2nd folate-dependent step in de novo purine biosynthesis, as its primary target. Consistent with targeting AICARFTase, using an in situ GARFTase assay in intact KB cells, inhibition by AG127 was modest and far less than for AG17 and AG23. Treatment of KB cells with AG127 resulted in time- and dose-dependent accumulation of the AICARFTase substrate ZMP (AICA ribonucleotide), confirming AICARFTase inhibition. ZMP accumulation was accompanied by activation of AMPK and inhibition of mTOR downstream targets. AG127 is a prototype of an entirely new generation of tumor-targeted antifolates with solid tumor selectivity over chemotherapy drugs currently available. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3822. doi:1538-7445.AM2012-3822

Abstract 3250: Design, synthesis and biological evaluation of side chain pemetrexed (PMX) analogues as potent inhibitors of tumors expressing folate receptors via GARFTase inhibition

Abstract Two major hurdles in cancer therapy are the inability of chemotherapy agents to selectively target tumor cells over normal tissues, and the development of tumor cell resistance. The multi-enzyme-targeted anticancer drug pemetrexed (PMX) suffers from dose-limiting toxicity due to its membrane transport by the reduced folate carrier (RFC) which is ubiquitously expressed in normal and tumor cells. We recently reported a series of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates. The most active of this series (AG71) included a 4 carbon bridge and was selectively transported into cells by folate receptors (FRs) and the proton-coupled folate transporter (PCFT) (but not RFC) whereupon it inhibited de novo purine biosynthesis at the level of glycinamide ribonucleotide formyltransferase (GARFTase). AG71 was highly active toward both KB and IGROV1 tumors; in SCID mice with KB tumors, AG71 was highly active against both early and advanced stage tumors. Based on the 5-substituted pyrrolo[2,3-d]pyrimidine structure of PMX and our results with the 6-substituted compound AG71, we synthesized 5-substituted pyrrolo[2,3-d]pyrimidine thienoyl analogs with 1 to 6 carbon atoms in the bridge (AG133-AG138) as hybrid molecules of PMX and AG71. The compounds were synthesized via a Heck coupling of the thiophene iodide with the alkene alcohols followed by α-bromonation and cyclization with the 2,4-diamino-6-oxo-pyrimidine to afford the 5-substituted pyrrolo[2,3-d]pyrimidine and elaborated to the L-glutamate. These analogs could be envisaged to afford the multi-targeted attributes of PMX, while preserving FRα and/or PCFT specificity of our 6-substituted analogs. Analogs were tested for anti-proliferative effects on KB human tumor cells and on Chinese hamster ovary (CHO) sublines engineered to individually express RFC (PC43-10), PCFT (R2/PCFT4), and FRα (RT-16). Analogs with 4 and 5-carbon atom chains (AG136 and AG137) showed IC50 values of 17.1 nM and 18.1 nM, respectively, against KB tumor cells in culture; other analogs of this series were 13- to >58-fold less active. In the CHO sublines, AG136 and AG137 were growth inhibitory in the order RT-16 = PC43-10 > R2/PCFT4 and RT-16 >> PC43-10 = R2/PCFT4, respectively. Thus, at least for AG136 unlike AG71, significant RFC activity is preserved. Whereas PMX is a multi-targeted agent with inhibitory effects on both de novo thymidylate and purine nucleotide biosynthetic pathways, the anti-proliferative effects of AG136 and AG137 were abolished by adenosine but not thymidine, establishing exclusive inhibition of purine biosynthesis. Our results suggest that 6-subtitution of the pyrrolo[2,3-d]pyrimidine scaffold is more effective in conferring selective transport of FRα over RFC, although inhibition of de novo purine biosynthesis is preserved in the 5-substituted compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3250. doi:10.1158/1538-7445.AM2011-3250

Abstract 4542: The design, synthesis and in vitro evaluation of selective FR α and β and PCFT substrates with potent GARFTase inhibitory activity as antitumor agents

Abstract Three folate transporters, the reduced folate carrier (RFC), folate receptors (FR) and the proton-coupled folate transporter (PCFT) account for most of the folate influx activity in mammalian cells. Lack of selectivity for folate transporters is one of the major causes of chemotherapy failure with antifolate drugs. We recently reported the potent glycinamideribonucleotide formyltransferase (GARFTase) inhibitory activity of a series of classical 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines with a thienoyl for benzoyl-substituted side chain with FR and PCFT specificity over RFC. In an attempt to optimize for selective transport by FR and PCFT, and GARFTase inhibitory activity, a furan for benzoyl-substituted side chain analog with a 4-carbon bridge was synthesized. The target compound was tested against KB human tumor cells that express FR alpha, human RFC, and human PCFT using a fluorescence-based cytotoxicity screen, and showed remarkably potent subnanomolar inhibitory activity (IC50 of 0.29 nM). The furan analog was further tested in isogenic Chinese hamster ovary sublines, engineered to express human FR alpha (RT-16), FR beta (D4), hRFC (pC43-10), or hPCFT (R2/hPCFT4). The furan side chain compound again showed a subnanomolar IC50s for both FR alpha and FR beta expressing cells (IC50 of 0.16 nM for RT16, and 0.92 nM for D4). Thus, in comparison with the lead thienoyl side chain compound (IC50 of 2 nM for RT16, and 0.75 nM for D4), the furan analog exhibited similar activities toward FR beta, but a 12.5-fold increased activity toward FR alpha. For PCFT-expressing R2/hPCFT4 cells, the furan side chain compound (IC50 of 439 nM) was about 10-fold less potent than the thienoyl analog (IC50 of 43.4 nM). The furan analog was inactive against human dihydrofolate reductase (hDHFR) or human thymidylate synthase (hTS) (IC50 >10−5 M), suggesting that neither hDHFR nor hTS is a principal target. Metabolite protection from in vitro cytotoxicity in KB cells showed that thymidine (10 μM) did not affect the growth inhibitory effects of the furan analog whereas adenosine (60 μM) and 5-amino-4-imidazolecarboxamide (320 μM) were completely protective, identifying GARFTase as the likely intracellular target. Our results indicate that the side chain heterocyclic ring may control the transport selectivity for both FR and PCFT of the pyrrolo[2,3-d]pyrimidines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4542.

Transient overexpression of κ and μ opioid receptors using recombinant adenovirus vectors

In order to study the trafficking and signal transduction mechanisms of the multiple opioid receptors, these receptors are expressed either transiently or stably in cell lines. Often, it is difficult to express receptors at a sufficiently high density to obtain reproducible results. To achieve a high density of receptors, replication-defective adenovirus (rAd5) vectors encoding the μ (MOR) and κ (KOR) opioid receptors, both in their native form and as fusion proteins bearing the green fluorescent protein (GFP) at their C-terminus, were constructed. These vectors efficiently and reproducibly infected Chinese hamster ovary (CHO) cells that stably express the human coxsackie–adenovirus receptor (hCAR), with up to 90% of cells becoming infected at a low multiplicity of infection (MOI). Saturation receptor binding studies using μ- and κ-selective agonists, [ 3 H ][ d-Ala 2, N-Me-Phe 4, Gly 5-ol]enkephalin (DAMGO) and [ 3 H ](5α7α,8β)-(−)- N-methyl- N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzeneacetamide (U69,593), respectively, and a nonselective antagonist, [ 3 H ]diprenorphine, revealed that rAd5-transduced cells expressed MOR and KOR for at least 3 days, at levels which exceeded those present on widely-used CHO sublines that stably express MOR or KOR. Expression levels were highest for the vectors encoding native MOR or KOR, and slightly reduced for the GFP fusion proteins. These findings demonstrate the feasibility of using rAd5 vectors to express opioid receptors at high densities, which may facilitate opioid receptor studies.

Cyclobutane Pyrimidine Dimers and Bulky Chemical DNA Adducts Are Efficiently Repaired in Both Strands of Either a Transcriptionally Active or Promoter-deleted APRT Gene

Both prokaryotic and eukaryotic cells have the capacity to repair DNA damage preferentially in the transcribed strand of actively expressed genes. However, we have found that several types of DNA damage, including cyclobutane pyrimidine dimers (CPDs) are repaired with equal efficiency in both the transcribed and nontranscribed strands of the adenine phosphoribosyltransferase (APRT) gene in Chinese hamster ovary cells. We further found that, in two mutant cell lines in which the entire APRT promoter region has been deleted, CPDs are still efficiently repaired in both strands of the promoterless APRT gene, even though neither strand appears to be transcribed. These results suggest that efficient repair of both strands at this locus does not require transcription of the APRT gene. We have also mapped CPD repair in exon 3 of the APRT gene in each cell line at single nucleotide resolution. Again, we found similar rates of CPD repair in both strands of the APRT gene domain in both APRT promoter-deletion mutants and their parental cell line. Our findings suggest that current models of transcription-coupled repair and global genomic repair may underestimate the importance of factors other than transcription in governing the efficiency of nucleotide excision repair.

Activation of Transfected M1or M3Muscarinic Acetylcholine Receptors Induces Cell–Cell Adhesion of Chinese Hamster Ovary Cells Expressing Endogenous Cadherins

Expression of endogenous cadherins by Chinese hamster ovary (CHO) cells has not been previously reported. However, we observed that CHO cells adhere to one another upon activation of transfected muscarinic acetylcholine receptors (mAChR), suggesting that the cells express endogenous cadherins. A 160-base pair RT-PCR product with 100% homology to the cytoplasmic domain of human E-cadherin was amplified from CHO cells. A second RT-PCR product amplified from these cells has 92% homology to the cytoplasmic domain of human cadherin-9 and 86% homology to the cytoplasmic domain of human cadherin-6. Western blotting indicates that CHO cells express a 165-kDa protein recognized by E-cadherin antibodies and a 120-kDa protein recognized by an antibody to the cadherin C-terminus sequence. The ability of transfected mAChR subtypes to regulate cadherin-mediated adhesion of CHO cells was tested by measuring the permeation of horseradish peroxidase across confluent CHO cell monolayers, by microscopic examination of the cells, and by aggregation assays. Cell–cell adhesion is induced within 15 min of activating transfected M1or M3mAChR which functionally couple to protein kinase C (PKC). In contrast, CHO cell adhesion is not affected by activating transfected M2mAChR which functionally couple to other effectors. Activation of PKC with phorbol esters also induces cell–cell adhesion of all CHO sublines tested. Immunofluorescence assays reveal that endogenous cadherins redistribute on the plasma membrane of CHO cells following mAChR or PKC activation. Inactivation of cadherins by removal of extracellular Ca2+abrogates adhesion induced by mAChR or PKC activation. Our demonstration that activation of only odd-numbered mAChR subtypes induces cadherin-mediated adhesion suggests that the unique responses of cells to M1or M3mAChR stimulation may involve cadherin activation.

Evidence of post-translational regulation of P-glycoprotein associated with the expression of a distinctive multiple drug-resistant phenotype in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells following exposure to fractionated X-irradiation in vitro dominantly expressed a distinctive multiple drug-resistant phenotype, characterised by resistance to vinca alkaloids, epipodophyllotoxins and colchicine, but not to anthracyclines, together with overexpression of P-glycoprotein (Pgp), but without any concomitant elevation in Pgp mRNA ( J Natl Cancer Inst 1990, 82, 607–612; 1992, 85, 48–53). To investigate the mechanism of this Pgp overexpression, Pgp stability was examined in an X-irradiation pretreated subline and compared with that of two colchicine-selected drug-resistant CHO sublines. These studies revealed a slower turnover of Pgp in the X-irradiated cells ( T 1 2 ≥ 40 h ) relative to the drug-selected sublines ( T 1 2 = 17 h ), indicating that Pgp overexpression appears to be differently regulated in these independently-derived resistant sublines. These data add support to our proposal that the development of drug resistance following X-irradiation may arise by a mechanism distinct from that operating after drug selection.

Protein glycosylation in heat-sensitive and thermotolerance-deficient mutants of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells are capable of developing a high degree of thermotolerance in response to appropriate heat conditioning. In this study we examined the relationship between thermotolerance development and protein glycosylation using four sublines of CHO cells. Two of these CHO sublines are characterized by an increased heat sensitivity and impaired cellular capacity for thermotolerance development. The data show that thermotolerance development after heat conditioning in the heat-sensitive, thermotolerance-deficient mutants was accompanied by reduced labeling of a Mr 50,000 glycoprotein (GP50), in both soluble and insoluble cell fractions. Similarly, activation of UDP-N-acetylgalactosaminyltransferase (Gal-NAcT) after hyperthermia was almost completely abolished in these cell lines. Both of these endpoints have been correlated previously with thermotolerance expression. The data are consistent with the glycosylation hypothesis that attributes increased heat resistance of thermotolerant cells, at least in part, to enhanced glycosylation and accumulation of endogenous glycoproteins, such as GP50.

DNA-mediated gene transfer in Chinese hamster ovary cells: clonal variation in transfer efficiency.

Thymidine kinase-deficient Chinese hamster ovary (CHO) cells were genetically transformed with the BamHI restriction fragment encoding the thymidine kinase gene of herpes simplex virus (HSV-tk). We have observed considerable clonal variation among independent CHO sublines with respect to transformation competence for the DNA-mediated gene transfer of HSV-tk. Transformation frequencies greater than or equal to 3 X 10(-4) were observed consistently in one subline, with a transformation efficiency of approximately 1 transformant per ng viral gene. The frequency and efficiency of transformation we observed in this system are at least 10-fold greater than those previously reported for DNA-mediated transformation of CHO cells by HSV-tk. All of the CHO HSV-tk+ transformants examined were stable for the transferred genotype in the absence of selection, and all showed evidence of co-transformation by unselected plasmid pBR322 sequences.