Hamster Cell Line Research Articles

Isolation of mutagen-sensitive Chinese hamster cell lines by replica plating.

Mutant rodent cell lines hypersensitive to DNA-damaging agents have provided a useful tool for the characterization of DNA repair pathways and have contributed to a better understanding of the mechanisms involved in the cellular responses to mutagenic treatment. Here we present a detailed description of how to isolate mutagen-sensitive mutants from hamster "wild-type" cell lines. First, cells are treated with ethyl nitrosourea, and then the mutagenized cell populations are screened for cells with an increased sensitivity to various mutagens using a replica-plating method. Mutagen-sensitive clones are identified and then characterized by assessing their stability, degree of sensitivity to various mutagens, and by genetic complementation analysis.

A Critical Role for DNA End-Joining Proteins in Both Lymphogenesis and Neurogenesis

XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.

Efficient PCNA complex formation is dependent upon both transcription coupled repair and genome overall repair

The protein proliferating cell nuclear antigen (PCNA) is an auxiliary factor for DNA polymerase δ and is involved in the resynthesis step of nucleotide excision repair (NER). After UV irradiation of quiescent cells, PCNA forms an insoluble complex with nuclear substructures. We have investigated associations between NER and its subcomponent pathway, transcription coupled repair (TCR) on PCNA complex formation using genetically related hamster cell lines with different repair characteristics. In DNA repair proficient cells, the PCNA complex was readily detectable within 30 min after UV irradiation by both immunofluorescence and western blot analyses. This complex formation after UV occurs efficiently in quiescent cells. In UV5 (human XP-D homolog) and UV 24 (human XP-B homolog) cells, which are totally deficient in NER, the PCNA complex was not detectable at 30 min after UV. The PCNA complex formation is restored to normal levels in UV5 cells after transfection with the human XPD gene, encoding a subunit of the basal transcription factor, TFIIH. In UV61 (Human CS-B homolog) cells, that are defective only in transcription coupled repair (TCR) of cyclobutane pyrimidine dimers (CPDs), the rate of PCNA complex formation was 2-fold slower than in repair proficient cells. This defect was complemented by transfection of the CSB gene into the UV61 cells. We thus conclude that efficient PCNA complex formation after UV is dependent upon both the NER and TCR pathways in hamster cells. The association of several other DNA repair proteins including XPA, RPA, TFIIH and p53 with the insoluble PCNA complex in UV treated cells suggests a central role for PCNA in different steps of NER.

The sensitization of cells treated with O6-methylguanine to alkylation damage is affected by the number of O6-methylguanine-DNA methyltransferase molecules escaped from inactivation

O6-Methylguanine (MeG) can bind to the active site of O6-methylguanine-DNA methyltransferase (MGMT) as a free base. The subsequent methyl transfer reaction inactivates the repair protein. Hence, MeG is used to deplete the active MGMT pools in Chinese hamster cell lines (CHO) transfected to express varying amounts of human MGMT. After treatment with the free base, a residual population of active protein molecules remains localized mostly in the cytoplasm. Depleted cells are then challenged with the alkylating drug mitozolomide. Genotoxicity of this agent varied among the cell lines, and the compound sensitivity seemed to be regulated by a steady state equilibrium of residual MGMT molecules between nucleus and cytoplasm.

Stable expression of human α-2,6-sialyltransferase in Chinese hamster ovary cells: functional consequences for human erythropoietin expression and bioactivity

Hamster cell lines are common hosts for recombinant protein production, e.g. erythropoietin (Epo). Terminal sialylation of native human proteins is characteristically in both α-2,3 and α-2,6 linkage to galactose at the termini of N-linked oligosaccharides but only in α-2,3 linkage in recombinant proteins expressed in hamster cells which do not express α-2,6-sialyltransferase (ST6GalI) (EC 2.4.99.1). This difference could alter the bioactivity of certain recombinant proteins. Chinese hamster ovary (CHO) cells stably transfected with human ST6GalI cDNA linked to the hamster metallothionein II promoter expressed highly inducible authentic ST6GalI activity. Untransfected CHO cells and CHO cells stably expressing ST6GalI cDNA when transfected with a human Epo cDNA expression cassette secreted immunoreactive Epo. Human Epo from singly transfected Pro -5 CHO cells induced significant reticulocytosis (7.00±1.58%; mean±S.D. % reticulocytes; control conditioned medium 3.04±1.29%; P<0.0024), whereas Epo from Pro -5 cells coexpressing ST6GalI elicited a more modest reticulocytosis (4.62±1.02%). Thus for recombinant human Epo, engineering CHO cells to express ST6GalI activity does not enhance Epo bioactivity in vivo in mice. The availability of CHO cells that express high levels of ST6GalI activity now enables systematic studies to determine the functional requirement for this form of sialylation in recombinant human proteins.

Spindle disturbances induced by benzo[ a]pyrene and 7,12-dimethylbenz[ a]anthracene in a Chinese hamster cell line (V79-MZ) and the stability of the numerical chromosome aberrations that follow

We previously reported that benzo[ a]pyrene (BP) and 7,12-dimethylbenz[ a]anthracene (DMBA) induce aneuploidy and polyploidy, respectively, in the Chinese hamster cell line V79-MZ in the absence of S9 mix. In the present study we investigated the effect of BP and DMBA on the mitotic spindle. BP caused incomplete spindle formation and DMBA inhibited spindle formation completely. The combined results indicate that incomplete spindles caused by BP resulted in aneuploidy, and the absence of spindle formation caused by DMBA resulted in polyploidy. The induced polyploidy was stable for several serial passages in fresh medium. BP and DMBA induced different chromosome number distributions. After BP treatment, the normal distribution of chromosome number was restored in 4 days. After DMBA treatment, on the other hand, a tetraploid peak was maintained for 2 months following an initial transient broad distribution of chromosome number after 1 day. The results suggest that different mechanisms were involved in the induction of numerical aberrations by BP and DMBA. Furthermore the induction of numerical aberrations by BP and DMBA was reproducible over 5 months of passages. In four clones tested, the frequency of cells with the modal chromosome number in control cultures gradually decreased from 82% on the average just after cloning to 62% 5 months later. BP and DMBA induced characteristic ploidy changes following succeeding cell passages for up to 5 months, indicating that the ability to respond to BP and DMBA was stable for that period of time. Because these findings were specific to V79-MZ cells, this cell line might be a good tool for studying chemicals that induce numerical chromosome aberrations.

Enhanced UV-induced mutagenesis in the UV61 cell line, the Chinese hamster homologue of Cockayne's syndrome B, is associated with defective transcription coupled repair of cyclobutane pyrimidine dimers

Cells from Cockayne's syndrome (CS) patients are hypersensitive to the cytotoxic effects of UV-irradiation and are defective in transcription coupled repair (TCR). We have examined the mutagenic consequences of impaired TCR in the Chinese hamster cell line UV61, the rodent homologue of CS complementation group B. Analysis of the two major UV-induced photolesions, cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PP), revealed that repair of CPD from the transcribed strand was strongly reduced in UV61 cells, but repair of 6-4 PP was indistinguishable from that in wild-type hamster cells. UV-induced mutation induction was enhanced in UV61 compared to that observed in repair proficient cells. The spectrum of UV-induced base substitutions in UV61 was clearly different from that observed in wild-type hamster cells and resembled the spectrum previously observed in nucleotide excision repair deficient hamster cells. In UV61 cells a strong strand bias for mutation induction was found; assuming that premutagenic lesions occur at dipyrimidine sequences, 76% of the mutations could be attributed to lesions in the transcribed strand. These data strongly favour the hypothesis that defective TCR of CPD is responsible for the enhanced UV-induced mutagenesis in UV61 cells.

Hypersensitivity of Ku-deficient cells toward the DNA topoisomerase II inhibitor ICRF-193 suggests a novel role for Ku antigen during the G2 and M phases of the cell cycle.

Ku antigen is a heterodimer, comprised of 86- and 70-kDa subunits, which binds preferentially to free DNA ends. Ku is associated with a catalytic subunit of 450 kDa in the DNA-dependent protein kinase (DNA-PK), which plays a crucial role in DNA double-strand break (DSB) repair and V(D)J recombination of immunoglobulin and T-cell receptor genes. We now demonstrate that Ku86 (86-kDa subunit)-deficient Chinese hamster cell lines are hypersensitive to ICRF-193, a DNA topoisomerase II inhibitor that does not produce DSB in DNA. Mutant cells were blocked in G2 at drug doses which had no effect on wild-type cells. Moreover, bypass of this G2 block by caffeine revealed defective chromosome condensation in Ku86-deficient cells. The hypersensitivity of Ku86-deficient cells toward ICRF-193 was not due to impaired in vitro decatenation activity or altered levels of DNA topoisomerase IIalpha or -beta. Rather, wild-type sensitivity was restored by transfection of a Ku86 expression plasmid into mutant cells. In contrast to cells deficient in the Ku86 subunit of DNA-PK, cells deficient in the catalytic subunit of the enzyme neither accumulated in G2/M nor displayed defective chromosome condensation at lower doses of ICRF-193 compared to wild-type cells. Our data suggests a novel role for Ku antigen in the G2 and M phases of the cell cycle, a role that is not related to its role in DNA-PK-dependent DNA repair.

Differential Stimulation of Cholesterol and Unsaturated Fatty Acid Biosynthesis in Cells Expressing Individual Nuclear Sterol Regulatory Element-binding Proteins

Three sterol regulatory element-binding proteins (SREBP-1a, -1c, and -2) stimulate transcription of genes involved in synthesis and receptor-mediated uptake of cholesterol and fatty acids. Here, we explore the individual roles of each SREBP by preparing lines of Chinese hamster ovary (CHO) cells that express graded amounts of nuclear forms of each SREBP (designated nSREBPs) under control of a muristerone-inducible nuclear receptor system. The parental hamster cell line (M19 cells) lacks its own nSREBPs, owing to a deletion in the gene encoding the Site-2 protease, which releases nSREBPs from cell membranes. By varying the concentration of muristerone, we obtained graded expression of individual nSREBPs in the range that restored lipid synthesis to near physiologic levels. The results show that nSREBP-2 produces a higher ratio of synthesis of cholesterol over fatty acids than does nSREBP-1a. This is due in part to a selective ability of low levels of nSREBP-2, but not nSREBP-1a, to activate the promoter for squalene synthase. nSREBP-1a and -2 both activate transcription of the genes encoding stearoyl-CoA desaturase-1 and -2, thereby markedly enhancing the production of monounsaturated fatty acids. nSREBP-1c was inactive in stimulating any transcription at the concentrations achieved in these studies. The current data support the emerging view that the nSREBPs act in complementary ways to modulate the lipid composition of cell membranes.

Identification of fragments of human transcripts froma defined chromosomal region: representational difference analysis of somatic cell hybrids.

We have tested representational difference analysis of cDNAs from somatic cell hybrids as a means to directly isolate expressed sequences derived from a defined chromosomal region. To this end, the hamster-human somatic cell hybrid Q1Z, which carries Xq28 as the only human chromosomal fragment, was used as Tester and the parental hamster cell line Y21 as Driver. After two rounds of subtraction, two major products of 510 and 307 bp were obtained, derived from the highly expressed human Xq28-derived QM gene and from a hamster repeat sequence strongly up-regulated in Q1Z, respectively. In a second subtraction experiment these fragments were added to the driver, to prevent their reappearance. After three rounds of subtraction a more complex difference product was obtained. Of 26 different fragments analysed, 12 fragments were derived from Xq28-derived genes, 10 of which were from known genes. One fragment was derived from a hamster gene strongly up-regulated in Q1Z. These results demonstrate that cDNA RDA can be used to isolate gene fragments from defined chromosomal regions and that suppression with major products, derived from highly expressed genes, is advantageous to isolate larger number of fragments, presumably derived from rarer transcripts.

CCG1/TAF(II)250 regulates epidermal growth factor receptor gene transcription in cell cycle mutant ts13.

The epidermal growth factor receptor (EGFR) plays a critical role in normal growth and its overexpression is associated with several types of cancer. To learn more about regulation of the expression of this important receptor, we investigated the role of the TAF(II)250 subunit of transcription factor IID in the transcription of the EGFR gene. The EGFR gene has a TATA-less promoter and TAF(II)250 has previously been shown to have an important regulatory role in such promoters. The study was performed in the ts13 hamster cell line which has a temperature-sensitive mutation in the CCG1 gene that encodes TAF(II)250. At the nonpermissive temperature, the transcription of a few cell cycle-dependent genes is depressed in ts13 cells while global RNA synthesis is unaffected. Using this model system, we found that EGFR promoter-driven luciferase expression in transiently transfected ts13 cells decreased 8, 25, and 50-fold after 12, 24, and 48 hours, respectively, at the nonpermissive temperature. The decrease was partially rescued by cotransfection with the wild-type CCG1 gene. The expression of endogenous EGFR also appeared to be regulated by TAF(II)250--the maximum binding capacity of ts13 cells for 125I-labeled EGF decreased approximately twofold when incubated for 2 days at the nonpermissive temperature. Placing these studies in the context of the current understanding of the TFIID transcription complex, we speculate that selective stimulation of EGFR gene transcription may be mediated by TAF(II)250 interaction with enhancer-bound activators and the basal transcription machinery.

UDP-glucose Deficiency Causes Hypersensitivity to the Cytotoxic Effect of Clostridium perfringens Phospholipase C

A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated. We now show that the mutant cell is 10(5) times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell. To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C. perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA. Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector. Exposure of these two types of transfectant clones to C. perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase. Further experiments with genetically engineered C. perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells.

Induction of anti-tumour immunity by suicide-gene-modified HPV-16-transformed hamster cells.

From K3/II, which is a highly oncogenic HPV16-transformed Syrian hamster cell line, thymidine-kinase(TK)-less cells, denoted B 49, were derived. B49 cells were transfected by a plasmid containing the herpes-simplex-virus TK gene (HSV TK) and several sub-lines expressing this gene were isolated from the transfected cultures. The HSV TK+ cells were highly sensitive to ganciclovir (GCV) and other anti-viral substances whose inhibitory effect is based on their phosphorylation by HSV TK. One of the cell lines, denoted KL1/6, exhibited relatively high stability of the HSV TK+ phenotype and was used in subsequent experiments. When KL1/6 cells were co-cultivated in the presence of GCV with various other cell lines of hamster, mouse or monkey origin, the by-stander effect (BE) was observed. GCV treatment of hamsters prevented development of tumours after the administration of KL1/6 cells but not K3/II cells. The treatment of animals with already established KL1/6-induced tumours resulted in tumour regression in all instances, but complete regression was observed only in animals carrying small tumours. The BE of KL1/6 cells on K3/II cells was also seen in vivo. In addition, concomitant immunity was observed in animals simultaneously inoculated with KL1/6 cells and K3/11 cells at 2 separate sites of the body. This effect was evident not only in animals in which KL1/6 tumours developed, but also in those in which tumour outgrowth was prevented by GCV treatment. In other experiments it was demonstrated that one KL1/6 + GCV treatment resulted in partial resistance, 2 such treatments in complete resistance to the challenge with K3/II cells.

Effects of nickel on DNA methyltransferase activity and genomic DNA methylation levels

Methylation of DNA plays an important role in organizing the genome into transcriptionally active and inactive zones. Nickel compounds cause chromatin condensation and DNA methylation in the transgenic gpt + Chinese hamster cell line (G12). Here we show that nickel is an inhibitor of cytosine 5-methyltransferase activity in vivo and in vitro. In living cells, this inhibition is transient and following a recovery period after nickel treatment, Mtase activity slightly rebounds. Genomic DNA methylation levels are also somewhat decreased following nickel treatment, but with time, there is an elevation of total DNA methylation above basal levels and before any rebound of methyltransferase activity. These results suggest that nickel exposure can elevate total genomic DNA methylation levels even when DNA methyltransferase activity is depressed. These findings may explain the hypermethylation of senescence and tumor suppressor genes found during nickel carcinogenesis and support the model of a direct effect of Ni 2+ on chromatin leading to de novo DNA methylation.

P48 Activates a UV-damaged-DNA binding factor and is defective in xeroderma pigmentosum group E cells that lack binding activity.

A subset of xeroderma pigmentosum (XP) group E cells lack a factor that binds to DNA damaged by UV radiation. This factor can be purified to homogeneity as p125, a 125-kDa polypeptide. However, when cDNA encoding p125 is translated in vitro, only a small fraction binds to UV-damaged DNA, suggesting that a second factor is required for the activation of p125. We discovered that most hamster cell lines expressed inactive p125, which was activated in somatic cell hybrids containing human chromosome region 11p11.2-11cen. This region excluded p125 but included p48, which encodes a 48-kDa polypeptide known to copurify with p125 under some conditions. Expression of human p48 activated p125 binding in hamster cells and increased p125 binding in human cells. No such effects were observed from expression of p48 containing single amino acid substitutions from XP group E cells that lacked binding activity, demonstrating that the p48 gene is defective in those cells. Activation of p125 occurred by a "hit-and-run" mechanism, since the presence of p48 was not required for subsequent binding. Nevertheless, p48 was capable of forming a complex with p125 either bound to UV-damaged DNA or in free solution. It is notable that hamster cells fail to efficiently repair cyclobutane pyrimidine dimers in nontranscribed DNA and fail to express p48, which contains a WD motif with homology to proteins that reorganize chromatin. We propose that p48 plays a role in repairing lesions that would otherwise remain inaccessible in nontranscribed chromatin.

Chromosomal Fragile Sites and DNA Amplification in Drug-resistant Cells

It has been well established that DNA amplification is one of the important mechanisms by which cultured cells acquire resistance to many cytotoxic compounds. Amplification of important genes including those encoding oncoproteins, growth factors, their receptors, and cell-cycle regulators has been reported in human neoplasms. Yet, despite intensive research since the first description of DNA amplification in cultured cells about 20 years ago, the mechanisms of DNA amplification remain largely unknown. Many models have been proposed to account for the diverse manifestations of amplified DNA in many different cell sources. It is not the intention of this commentary to review these many different models. Rather, we will focus on the recent advances in this area of research, made mainly via the fluorescence in situ hybridization technique, that have revealed a fairly common chromosomal manifestation of amplified DNA in the drug-resistant hamster cell lines and have demonstrated the association of chromosomal fragile site breakage with early events in DNA amplification. These new developments underscore the importance of future research toward understanding the molecular bases of chromosomal fragile sites, including mechanisms involved in DNA strand breakage and repair, chromosomal translocations, and deletions, which may, in turn, provide important new insights into genomic plasticity and neoplastic transformation.

Similar mutational spectra in the HPRT gene of human and hamster cell lines after exposure to either low dose rate or high dose rate X-rays

The dose rate at which cells are exposed to X-rays may influence the nature of induced mutations. To investigate this, the molecular spectra were determined at the HPRT gene in a hamster (V79) and a human (WI-L2-NS) cell line after the same total dose of X-rays has been administered at either a low dose rate (LDR; 3.33 mGy/min) or a high dose rate (HDR; 1.24–1.4 Gy/min) X-irradiation. Mutational spectra appeared similar, the fraction of mutants carrying deletions ranging between 59%–66% for the V79 strain and between 64%–75% for the WI-L2-NS strain, and independent of the irradiation conditions. The data indicate no effect of ongoing repair processes under LDR conditions on the kind of induced mutations in mammalian cells.

Ubiquitination Is Required for the Retro-translocation of a Short-lived Luminal Endoplasmic Reticulum Glycoprotein to the Cytosol for Degradation by the Proteasome

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded. Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cell fractionation experiments indicate that RI332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI332, its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61. From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.

The 1.7 A crystal structure of the regulator of chromosome condensation (RCC1) reveals a seven-bladed propeller.

The gene encoding the regulator of chromosome condensation (RCC1) was cloned by virtue of its ability to complement the temperature-sensitive phenotype of the hamster cell line tsBN2, which undergoes premature chromosome condensation or arrest in the G1 phase of the cell cycle at non-permissive temperatures. RCC1 homologues have been identified in many eukaryotes, including budding and fission yeast. Mutations in the gene affect pre-messenger RNA processing and transport, mating, initiation of mitosis and chromatin decondensation, suggesting that RCC1 is important in the control of nucleo-cytoplasmic transport and the cell cycle. Biochemically, RCC1 is a guanine-nucleotide-exchange factor for the nuclear Ras homologue Ran; it increases the dissociation of Ran-bound GDP by 10(5)-fold. It may also bind to DNAvia a protein-protein complex. Here we show that the structure of human RCC1, solved to 1.7-A resolution by X-ray crystallography, consists of a seven-bladed propeller formed from internal repeats of 51-68 residues per blade. The sequence and structure of the repeats differ from those of WD40-domain proteins, which also form seven-bladed propellers and include the beta-subunits of G proteins. The nature of the structure explains the consequences of a wide range of known mutations. The region of the protein that is involved in guanine-nucleotide exchange is located opposite the region that is thought to be involved in chromosome binding.

Specific Substitutions at Amino Acid 256 of the Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transport ATPase Mediate Resistance to Thapsigargin in Thapsigargin-resistant Hamster Cells

High levels of resistance to thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCAs), can be developed in culture by stepwise exposure of mammalian cells to increasing concentrations of TG. We have identified, in two independently selected TG-resistant hamster cell lines of different lineages, mutant forms of SERCA. In the TG-resistant Chinese hamster lung fibroblast cell line DC-3F/TG, a T --> C change at nucleotide 766 introduces a Phe256 --> Leu alteration within the first cytosolic loop of the SERCA. In contrast, in the TG-resistant Syrian hamster smooth muscle cell line DDT/TG 4 microM, a T --> C change at nucleotide 767 introduces a Phe256 --> Ser mutation at that position. When these specific mutations are introduced into a wild-type full-length avian SERCA1 cDNA, transfection experiments reveal that Ca2+ transport function and ATP hydrolytic activity are not altered by such mutations. However, a 4-5-fold resistance to TG inhibition of Ca2+ transport function occurs upon the introduction of either the Phe256 --> Leu or the Phe256 --> Ser mutation into wild-type SERCA1. These specific mutations also render the hydrolytic activity of the ATPase resistant to inhibition by TG. Our results not only implicate amino acid 256 in TG-SERCA interactions, but also demonstrate that specific mutations within SERCA can mediate resistance to TG.