Metabolic co-operation in HGPRT+ and HGPRT− embryonal carcinoma cells

HGPRT⁺ and HGPRT^? clones have been isolated from the mouse embryonal carcinoma tissue culture line PC13. Upon inoculation into isogeneic mice, all clones form tumors showing the same pattern of differentiation as that given by PC13, namely, a predominance of neural differentiation with the presence of smaller amounts of other tissues. The karyotype of one HGPRT clone, PC13TG8, has been compared with that of PC13 by G-banding. Both lines possess a marker isochromosome and show a substantial degree of chromosome imbalance. By two techniques, (a) autoradiography after [³H]hypoxanthine labelling and (b) co-culture in toxic purine analogues, PC13 and PC13TG8 cells have been shown to participate in metabolic co-operation with each other and with the Chinese hamster cell line Don and its derivatives. Like other tissue culture lines, however, they show little or no metabolic co-operation with derivatives of mouse L cells.     

Evidence for linkage between glucose 6-phosphate dehydrogenase and hypoxanthine-guanine-phosphoribosyl transferase loci in Chinese hamster cells

G6PD and 6PGD activities were determined in diploid, hyperdiploid, tetraploid, and hybrid cells all originating from the same Chinese hamster cell line (the DON line). A relationship between gene multiplicity and enzyme activity has been observed. The same enzymes were studied in hybrid cells cultivated in selective media. Selection was carried out against and for the HGPRT⁺ locus. The differences in G6PD and 6PGD activities between the cell lines found under these conditions indicate a positive linkage of the G6PD and HGPRT loci and negative linkage of the 6PGD and HGPRT loci in these Chinese hamster cells.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

Microscale isoelectric focusing studies of mouse and human hypoxanthine-guanine phosphoribosyl transferases

A microscale isoelectric focusing technique has been developed and used to study hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C. 2.4.2.8, inosinate-guanylate:pyrophosphate phosphoribosyl transferase) activities in mouse and human cell lines. The enzymes of both mouse and human origin are shown to exhibit considerable heterogeneity, but each type has a unique range of isoelectric pH. The enzyme of a mouse × human hybrid cell line, derived by fusion of HGPRT^– parental cells, gives a homogeneous peak of activity, unlike the wild-type enzyme of either parent. The possibility is suggested that this enzyme activity is due to intra-allelic complementation.Centennial Fellow of the Medical Research Council of Canada, 1967–1970.     

Reaction of antibody to normal human hypoxanthine phosphoribosyltransferase with products of mutant genes.

Immunoglobulin produced in rabbits against normal human red cell hypoxanthine phosphoribosyl transferase (HPRT, EC 2.4.2.8) was used to study cell lysates of individuals with deficient enzyme activity. The reaction of immunoglobulin with HPRT formed partially active insoluble and fully active soluble complexes. The insoluble complexes were separated from soluble complexes and the free enzyme by centrifugation. The soluble complexes and free enzyme were separated by electrophoresis. Hemolysates from 13 patients with the Lesch-Nyhan syndrome who have virtually total deficiency of HPRT activity and 2 patients with hyperuricemia and 2–5% of normal activity were unable to neutralize immunoglobulin and showed no evidence of cross-reacting material (CRM). In contrast, 2 other partially deficient males with 4.5 and 50% of normal actvity, and a partially deficient heterozygous female with 34% of normal activity, were CRM⁺ in this assay. The amount of CRM present in the cells of these 2 males appeared to be disproportionate to their HPRT activity. The heterozygous female contained about 30% of normal CRM which was consistent with the estimated activity provided by her normal cell population. This indicated that her abnormal cells were CRM^?. Absence of CRM in her abnormal cells was consistent with the observed lack of CRM in hemolysates of her hyperuricemic half-brother. These data indicate the presence of considerable heterogeneity in human mutation at the HPRT locus.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing aminopterin

When seeded in small numbers in medium containing 10^?6M aminopterin and fetal calf serum, V₇₉ Chinese hamster cells required dialyzable components from the serum for growth. However, the cells grew in medium containing 10^?6M aminopterin and dialyzed serum, provided that the medium was supplemented with 10^?5M hypoxanthine and sufficient 5·10^?6M) thymidine. A growth-inhibitory property of some batches of dialyzed serum was abolished on heating the serum for 30 min at 56°. Three lines of V₇₉ cells which lacked detectable hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity were seleccted in medium containing 8-azaguanine (8-AzG). In two of these, no spontaneous reversion to the HGPRT⁺ phenotype was detectable, and these cells did not cooperate metabolically with HGPRT⁺ cells to prevent the growth of the latter in HAT medium. One of the HGPRT^? lines showed a high rate of spontaneous reversion (118/10⁵ cells) in medium containing undialyzed serum. However, in medium containing dialyzed serum the spontaneous reversion rate fell to $\text{4}\text{10}{}^5\text{cells}$, suggesting that the revertants arising in medium containing undialyzed serum were biochemically heterogeneous.     

The Characterization of α-Glycerophosphate Dehydrogenase Mutants in DROSOPHILA MELANOGASTER

Thirty mutants of α-glycerophosphate dehydrogenase (αGPDH, EC 1.1.1.8) from Drosophila melanogaster were produced with the chemical mutagen ethyl methanesulfonate (EMS). These mutants and nine others previously obtained have been characterized with respect to level of enzymatic activity, viability, flight ability, and presence of cross-reacting material (CRM). The presence of αGPDH mRNA in several of the mutants has been tested by in vitro translation. There are strong correlations between the level of enzyme activity, viability and flight ability. Thirteen of the mutants are CRM^- by solution immunoprecipitation experiments, but of these, only three are CRM^- by a more sensitive ¹²⁵ I-protein A-based radioimmune gel assay. The viability of the three CRM^- mutants suggests that the absence of αGPDH protein is not a lethal condition. The immunoprecipitated protein of the low activity mutant, αGpdh^nGL3, has a smaller apparent molecular weight on polyacrylamide-SDS gels than does the protein from wild type. Criteria for the identification of nonsense mutations in Drosophila are discussed.     

应用RNA干扰技术体外抑制兔成纤维细胞HGPRT的表达及其核移植研究

次黄嘌呤-鸟嘌呤磷酸核糖基转移酶( hypoxanthine guanine phosphoribosyltransferase,HGPRT )的功能缺失与痛风、肾结石和雷纳综合症(Lesch-Nyhan Syndrome)等疾病相关．制作HGPRT基因表达降低的模式动物,将有利于人们对这种疾病的发病机理和治疗做进一步的研究．构建了针对HGPRT基因表达的shRNA干扰载体,并将质粒转染兔成纤维细胞,获得携带该干扰片段的转基因细胞系,经PCR鉴定转基因成纤维细胞克隆阳性率为83.3%．RT-PCR及Western blot检测结果表明转基因干扰成纤维细胞系HGPRT mRNA和蛋白质表达量明显降低．最后,以转基因成纤维细胞进行核移植,囊胚率为27.8%,与正常来源的成纤维细胞囊胚率相比较差异不显著．说明,通过RNAi可稳定干扰兔成纤维细胞HGPRT基因的表达,为进一步通过核移植技术建立HGPRT RNAi转基因兔模型创造条件．     

Rapid determination of hypoxanthine-guanine-phosphoribosyl transferase in human fibroblasts and amniotic cells

Summary A micromodification of the method of HGPRT and APRT assay is described, which measures the incorporation of ¹⁴C hypoxanthine and ¹⁴C adenine into cultured skin fibroblasts and amniotic cells grown on microtiter plates. Only about 10 000 cells are needed per assay. By this method HGPRT deficient cells can be easily distinguished from normal cells. Investigations with respect to the effect of substrate concentrations and time of incubation have been carried out on some normal fibroblast cell lines, amniotic cell lines and 3 Lesch-Nyhan cell lines.Another modified method is described for quantitative determination of HGPRT activity by means of radio thin-layer chromatography.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Synthesis of purines in human lymphoblast cells deficient in methylthioadenosine phosphorylase activity

Two human lymphoblastic cell lines, deficient in methylthioadenosine phosphorylase (MTAP) activity, were found to have increased rates of de novo purine synthesis. These MTAP⁻ cell lines were K562, an undifferentiated leukemic line and CCRF-CEM, a leukemic line of T-cell origin. Another T-cell line, CCRF-HSB-2 was found to be deficient in activity. However, this line did not demonstrate elevated rates of purine synthesis. Purine metabolism in the above cell cultures was compared with MTAP⁺ human B-cell lines and two human T-cell lines (MOLT-3 and MOLT-4). In all the MTAP⁺ cell lines, the rate of de novo purine synthesis was inhibited by the presence of methylthioadenosine in the assay medium (10 μM concentration produced more than 90% inhibition). However, purine synthesis in the MTAP⁻ cells was resistant to inhibition by methylthioadenosine. Adenine in the assay medium inhibited de novo purine synthesis in MTAP⁺ and MTAP⁻ cells to a similar degree. This inhibition was dose dependent and was elicited by concentrations similar to those of methylthioadenosine. Growth of the cell lines in culture was not affected by either methylthioadenosine or adenine at the concentrations which produced inhibition of purine synthesis. These results suggest that purine synthesis in MTAP⁺ cells is inhibited by adenine formed from the phosphorolytic cleavage of methylthioadenosine by methylthioadenosine phosphorylase.     

Mechanism of cytotoxic action of azaguanine and thioguanine in wild-type V79 cell lines and their relative efficiency in selection of structural gene mutants

The cytotoxic effects of azaguanine and thioguanine have been compared in two wild-type V79 cells. To achieve equitoxic effects in both cell lines a 10–20-fold higher concentration of azaguanine than thioguanine was required. Affinity of HGPRT for azaguanine was 10-fold lower than for hypoxanthine in both cell lines and was similar to that for thioguanine in V79S cells. Affinity for thioguanine differed by a factor of 3 in the two cell lines. The rate of cell kill by azaguanine was markedly slower than by thioguanine in both cell lines. Reduction of whole cell uptake of [¹⁴C]hypoxanthine incorporation by unlabelled azaguanine was only demonstrable after prolonged incubation periods as was incorporation of [¹⁴C]azaguanine into acid-insoluble material. Experiments with cell-free extracts indicated that hypoxanthine acts as a non-competitive inhibitor of the enzyme. The slow rate of dissociation of the HGPRT—azaguanine complex is reflected in the slow rate of killing of wild-type cells. Clones resistant to the cytotoxic effects of these analogues have been selected from both cell lines and have been shown to possess HGPRT with altered kinetic properties. Our data suggest that azaguanine and thioguanine may select for mutations at different sites on the HGPRT molecule in V79 cells and provide possible explanations for the differences in effectiveness of these two agents reported in other cell lines.     

Activation of low and null activity isozymes of maize alcohol dehydrogenase by antibodies

Summary Antisera were raised against several purified, high specific acitivity isozymes of maize alcohol dehydrogenase (ADH1). The various antisera had different effects on the activity of immunoprecipitated ADH. One antiserum completely inactivated maize ADH. This inactivation could be blocked by preicubation of the enzyme with NAD⁺, its cofactor, or with NADP. The different antisera were used to analyze variant froms of ADH1. Isozymes having lowered specific activity were activated to wild-type levels by precipitation of the enzymes with noninactivating antisera. Isozymes having no detectable ADH activity (CRM⁺ nulls) were activated by immunoprecipition with noninactivating antisera when preincubated with NAD⁺ or NADP. All of the CRM⁺ nulls were shown to be unable to bind NAD⁺, a flaw which can account for their lack of activity. The results indicate that a conformational equilibrium between active and inactive forms of maize ADH in solution controls the specific activity of the various isozymes. Both controls the specific activity of the various isozymes. Both NAD⁺ and antibodies raised against high specific activity enzymes can interact with low activity isozymes to shift the balance of the equilibrium toward the active form, thus increasing their specific activity.     

Chromosome-mediated transfer of murine alleles for hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and ouabain resistance into human cell lines

Genetic drug-resistance markers were transferred via purified metaphase chromosomes from mouse L cells into the human fibrosarcoma line HT1080 and HeLa S3 cells. Interspecific chromosome-mediated transfer of hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC 2.4.2.8) from mouse L cells into HGPRT^– HT1080 cells occurred at a frequency of approximately 1×10^–7. The presence of the mouse allele for HGPRT in transferent isolates was confirmed by isoelectric focusing. Transfer of ouabain resistance from mouse L cells to HT1080 and HeLa S3 cells occurred at an average frequency of approximately 4×10^–7. Expression of the mouse trait in transferent isolates was confirmed by their ability to withstand doses of ouabain which would be lethal to spontaneous ouabain-resistant mutants of the human cells but not to mouse L cells. Ouabain-resistant transferents of human cells showed 10⁴- to >10⁵-fold enhanced drug resistance, characteristic of either wild-type or mutant alleles, respectively, from ouabain-resistant donor L cells. Unstable expression of the transferred phenotypes in the absence of selection was seen in some isolates, but expression was lost at slow rates.This work was supported by National Institutes of Health Grant GM30383/21665 to RMB, Core Grants CA14051 to S. E. Luria and CA24538 to E. Mihich, and institutional predoctoral Training Grant GM07287.     

Identification of cancer stem cell markers in human malignant mesothelioma cells

Malignant mesothelioma (MM) is an aggressive and therapy-resistant neoplasm arising from the pleural mesothelial cells and usually associated with long-term asbestos exposure. Recent studies suggest that tumors contain cancer stem cells (CSCs) and their stem cell characteristics are thought to confer therapy-resistance. However, whether MM cell has any stem cell characteristics is not known. To understand the molecular basis of MM, we first performed serial transplantation of surgical samples into NOD/SCID mice and established new cell lines. Next, we performed marker analysis of the MM cell lines and found that many of them contain SP cells and expressed several putative CSC markers such as CD9, CD24, and CD26. Interestingly, expression of CD26 closely correlated with that of CD24 in some cases. Sorting and culture assay revealed that SP and CD24⁺ cells proliferated by asymmetric cell division-like manner. In addition, CD9⁺ and CD24⁺ cells have higher potential to generate spheroid colony than negative cells in the stem cell medium. Moreover, these marker-positive cells have clear tendency to generate larger tumors in mouse transplantation assay. Taken together, our data suggest that SP, CD9, CD24, and CD26 are CSC markers of MM and could be used as novel therapeutic targets.     

Reversion in expression of hypoxanthine-guanine phosphoribosyltransferase in 6-thioguanine resistant neuroblastoma: evidence for reduced enzyme levels associated with unaltered catalytic activity.

Five clones of mouse neuroblastoma cells able to grow in hypoxanthine-aminopterin-thymidine containing medium were isolated from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8; IMP: pyrophosphate phosphoribosyltransferase) deficient cell line. These hypoxanthine-aminopterin-thymidine resistant revertant clone had 45-55% of wild-type cell HGPRT activity. Kinetic studies indicated that the HGPRT in revertant clones had a reduced maximal velocity as compared to wild type cells based on cell protein. Apparent Km values of HGPRT for hypoxanthine and 5-phosphoribosyl-1-pyrophosphate were similar in wild-type and revertant cells. Heat inactivation studies demonstrated a similar heat lability for HGPRT in revertant and wild-type cells. An antibody fraction prepared from serum of rabbits immunized with HGPRT partially purified from mouse liver was used to measure the amount of cross-reacting material in normal and revertant clones. The revertant clones had one-half the amounth of cross-reacting material present in wild-type cells, based on a given amount of cell protein. These data indicate that the revertant cells may contain fewer HGPRT molecules with unaltered catalytic activity.     

Cytogenetics and the major histocompatibility complex in a proliferating microcell-mediated hybrid clone

Summary By way of a microcell fusion, three chromosomes from a B82HTQ2 (TK^–) cell were introduced into a PG19 (HGPRT^–) cell. Analysis of this hybrid clone showed that the transferred chromosomes restored a positive HGPRT status but failed to produce heterozygosity for the major histocompatibility complex (H-2). The three chromosomes also proved stable in both long term culture in vitro and tumor testing in mice. It is suggested that the method could prove useful in correcting genetic defects or in introducing new genetic characteristics without the introduction of the genes coding for major histocompatibility antigens. The surface structure of the microcells was studied by scanning electron microscope. The optimum for induction of the microcells from B82HTQ2 cells and its' purification were reported here. Frequency of the sister chromatid exchange (SCE) of the hybrid cells and their sensitivity to mitomycin C (MMC) were also examined.     

Interference in hybrid clone selection caused by Mycoplasma hyorhinis infection

Mycoplasma hyorhinis strains were isolated from Chinese hamster DON cells which lacked the ability to produce hybrid colonies in HAT medium. The mycoplasma isolates were virtually devoid of HGPRT activity in vivo and in vitro in the presence of excess co-enzyme, phosphoribosylpyrophosphate. Deliberate infection of mycoplasma-free cells caused no alterations in the HGPRT^? and TK^? phenotypes of the cells. Heterokaryon formation with infected cells was normal and the failure to produce hybrid colonies resulted from depletion, by nucleoside phosphorylase activity, of exogenous thymidine required for rescue of hybrid cells in HAT medium. Increasing the thymidine concentration and repeatedly replenishing HAT medium permitted hybrid clone formation.     

Analysis of HGPRT- CRM+ human lymphoblast mutants.

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.     

Cell surface antigens of clonal teratocarcinoma cells at various stages of differentiation

We have studied cell surface antigen expression of teratocarcinoma cells at various stages of differentiation. These cells can be maintained in the undifferentiated state or will differentiate in vitro in a manner which parallels the early development of the mouse embryo. Three antigens were studied: a stem cell antigen (C); the major histocompatibility alloantigens (H-2); and the alloantigen Thy-1.The stem cell antigen was recognized by an anti-serum raised against a pluripotent teratocarcinoma cell line. This antiserum was shown to label embryonal carcinoma cells and early mouse embryo cells. The activity of the antiserum against embryonal carcinoma cells could be adsorbed with brain, kidney, and sperm from adult mice.The phenotype of the undifferentiated embryonal carcinoma cells is C⁺, H-2⁻, Thy-1⁻ or C⁻, H-2⁻, Thy-1⁻. The first stage in the process of differentiation is the formation of simple embryoid bodies with a layer of endodermal cells surrounding an inner core of embryonal carcinoma cells. The endodermal cells are C⁻, H-2⁻, Thy-1⁻. Further differentiation of the embryoid bodies attached to a substratum is associated with the appearance of H-2⁺ and Thy-1⁺ cells in the cultures.     

Ferulic acid maintains the self-renewal capacity of embryo stem cells and adipose-derived mesenchymal stem cells in high fat diet-induced obese mice

Self-renewal is required for embryo stem cells (ESCs) and adipose-derived mesenchymal stem cells (ADMSCs). This study examined the ability of ferulic acid in mouse ESCs and ADMSCs, in a high fat diet-induced mouse model. Initially, five natural compounds of ferulic acid, xanthohumol, curcumin, ascorbic acid, and quercetin were screened in ESCs using an alkaline phosphate ⁺(AP⁺) assay, as a self-renewal biomarker. A ferulic acid treatment was the highest AP⁺ staining in hop-hit screening compounds. Also a ferulic acid increased Nanog mRNA levels in ESCs. The in vivo effects of ferulic acid were next examined in an obese mouse model. C57BL/6 J male mice were fed either a high fat diet (HFD) or control diet with ferulic acid (5 g/kg diet) for 8 weeks. Ferulic acid exhibited weight loss and improved glucose homeostasis, lipid profiling, and hepatic steatosis in a HFD-induced mouse model. Next, ADMSCs (Sca-1⁺CD45⁻), a hallmark of fat stem cells, were then isolated and quantified from mouse abdominal adipose tissue. A HFD decreased the Sca-1⁺CD45⁻ cell population of ADMSCs, but HFD-induced obese mice given ferulic acid showed an increased the Sca-1⁺CD45⁻ cell population of ADMSCs. Moreover, ferulic acid enhanced NANOG mRNA levels in human ADMSCs and its related gene mRNA expression. Overall, this study suggests that ferulic acid preserves self-renewal in ESCs, and contributes to ADMSCs self-renewal and effective weight control in obesity.