Differential formation and enhanced removal of specific cisplatin-DNA adducts in two cisplatin-selected resistant human testicular teratoma sublines

Mechanisms of cisplatin resistance have been studied in two independently-selected sublines expressing clinically-relevant levels of resistance (3-fold) and established from a primary testicular teratoma obtained from previously untreated patients. Resistance was not associated with any significant modification in cellular uptake of cisplatin, in total glutathione levels or associated enzyme activities. However, immunochemical quantitation of specific platinum-DNA adduct formation and removal revealed that both resistant sublines were more proficient in repairing certain adducts than their generally repair deficient respective parental lines. SUSA/CP+ cells were more efficient in removing the intrastrand adducts in the sequence Pt-AG and the bi-functional Pt-(GMP)2 lesions, as well as DNA-DNA interstrand cross-links, whilst H12.1/DDP cells were highly proficient in removing the major Pt-GG intrastrand adducts.     

Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell

A technique for the isolation of gamma ray-sensitive Chinese hamster ovary (CHO) cell mutants is described, which uses nylon cloth replica plating and photography with dark-field illumination to directly monitor colonies for growth after gamma irradiation. Two gamma ray-sensitive mutants were isolated using this method. One of these cells (XR-1) had a two-slope survival curve: an initial steep slope and then a flattening of the curve at about 10% survival. Subsequently, it was found that this cell is sensitive to gamma irradiation in G1, early S, and late G2 phases of the cell cycle, whereas in the resistant phase (late S phase) its survival approaches that of the parental cells. The D37 in the sensitive G1 period is approximately 30 rads, compared with 300 rads of the parental cell. This mutant cell is also sensitive to killing by the DNA breaking agent, bleomycin, but is relatively insensitive to UV light and ethyl methane sulfonate, suggesting that the defect is specific for agents that produce DNA strand breakage.     

Glycosidase activities in Chinese hamster ovary cell lysate and cell culture supernatant

To probe the potential for extracellular degradation of glycoprotein oligosaccharides in conjunction with Chinese hamster ovary (CHO) cell culture, an initial characterization of several CHO cell glycosidases was performed using 4-methylumbelliferyl substrates. CHO cell lysates contained sialidase, beta-galactosidase, beta-hexosaminidase, and fucosidase activities with pH optimums near 5.5, 4, 6, and 6.5, respectively. These glycosidase activities were also present in cell-free supernatant samples from commercial CHO cell cultures. The sialidase activity was further characterized. In contrast to previous reports concerning mammalian sialidases, the sialidase activity in CHO cell lysate retained considerable activity at pH 7 and was very stable, with a half-life of 57 h at 37 degrees C. Both the Km and Vmax of CHO lysate sialidase for 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4MU-NeuAc) varied with pH, and this activity was competitively inhibited by 2,3-dehydro-2-deoxy-N-acetylneuraminic acid and by free N-acetylneuraminic acid. The kinetic characteristics and pH-activity profiles of the CHO cell lysate and cell culture supernatant sialidase activities were essentially identical, and both released sialic acid from the glycoprotein fetuin at pH 7.5. These results suggest that the oligosaccharides of glycoproteins secreted by CHO cells can potentially be modified extracellularly by sialidase under culture conditions which promote the release and extracellular accumulation of this enzyme.     

Gene silencing by DNA methylation and dual inheritance in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells strain D422, which has one copy of the adenine phosphoribosyl transferase (APRT) gene, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate. Cells with a silenced APRT gene were selected on 2, 6-diaminopurine. Colonies were isolated and shown to be reactivated to APRT+ by 5-aza-cytidine and by selection in medium containing adenine, aminopterin and thymidine. Genomic DNA was prepared from eight isolates of independent origin and subjected to bisulphite treatment. This deaminates cytosine to uracil in single-stranded DNA but does not deaminate 5-methyl cytosine. PCR, cloning and sequencing revealed the methylation pattern of CpG doublets in the promoter region of the APRT- gene, whereas the active APRT gene had nonmethylated DNA. CHO strain K1, which has two copies of the APRT+ gene, could also be silenced by the same procedure but at a lower frequency. The availability of the 5-methyl dCTP-induced silencing, 5-aza-CR and a standard mutagen, ethyl methane sulphonate, makes it possible to follow concomitantly the inheritance of active, mutant or silenced gene copies. This analysis demonstrates "dual inheritance" at the APRT locus in CHO cells.     

Sensitivity of a mutator gene in Chinese hamster ovary cell to deoxynucleoside triphosphate pool alterations

The Thy- mutants of Chinese hamster ovary cells have a 5- to 10-fold elevated pool of deoxycytidine 5'-triphosphate (dCTP) and are auxotrophic for thymidine as an apparent consequence of a single mutation. thy is also a mutator gene, elevating the spontaneous rate of mutation 5- to 200-fold for at least two genetic markers. Previous experiments suggested that this mutator activity was caused by the elevated pool of dCTP in Thy- cells. To test this, the dCTP and deoxythymidine 5'-triphosphate (dTTP) pools were manipulated by altering the external concentration of thymidine in the growth medium. The rate of mutation at one genetic locus, ouabain resistance, was directly related to cellular dCTP content. At the highest level of dCTP the rate in one Thy- strain was approximately 200 times that of wild-type cells. However, the relationship between dCTP content and the rate of mutation at the ouabain locus was different for two mutator strains and wild-type cells. The rate of mutation at a second locus, thioguanine resistance, was increased approximately 10-fold over wild type regardless of the dCTP-dTTP pools. These experiments suggest that the mutator activity of thy is clearly related to dCTP content, but the dCTP level alone does not appear to be the cause of the mutator.     

A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants

Chinese hamster ovary cells selected for resistance to colchicine display pleiotropic cross-resistance to a wide range of amphiphilic drugs. The drug-resistant phenotype is due to a membrane alteration which reduces the rate of drug permeation. Surface labelling studies reveal that drug-resistant Chinese hamster ovary cell membranes possess a carbohydrate-containing component of 170 000 daltons apparent molecular weight which is not observed in wild type cells. Through studies of the metabolic incorporation of carbohydrate and protein precursors, and through the use of selective proteolysis, this component is shown to be a cell surface glycoprotein. Since this glycoprotein appears unique to mutant cells displaying altered drug permeability, we have designated it the P glycoprotein. The relative amount of surface labelled P glycoprotein correlates with the degree of drug resistance in a number of independent mutant and revertant clones. A similar high molecular weight glycoprotein is also present in drug-resistant mutants from another hamster cell line. Observations on the molecular basis of pleiotropic drug resistance are interpreted in terms of a model wherein certain surface glycoproteins control drug permeation by modulating the properties of hydrophobic membrane regions...     

Mitochondrial dysfunction of a cultured Chinese hamster ovary cell mutant deficient in cardiolipin

In our preceding paper, we reported that a temperature-sensitive Chinese hamster ovary cell mutant, PGS-S, with thermolabile phosphatidylglycerophosphate synthase was defective in the biogenesis of both phosphatidylglycerol and cardiolipin (CL) at a nonpermissive temperature (Ohtsuka, T., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 22908-22913). To investigate the biological role of cardiolipin, we examined the structure and function of mitochondria in mutant PGS-S cells, since CL is primarily found in the mitochondrial membranes of eukaryotic cells. Under conditions where the formation of CL was impaired, this mutant had both morphological and functional mitochondrial abnormalities, manifested by more stringent temperature sensitivity for cell growth in glucose-deficient medium and by reduced ATP production, increased glycolysis, and reduced oxygen consumption in intact cells. Rotenone-sensitive NADH oxidase activity in cell extracts was also reduced in the mutant cultivated at a nonpermissive temperature, showing a defect(s) in the respiratory electron transport chain of mitochondria. Of the respiratory chain complexes, rotenone-sensitive NADH-ubiquinone reductase (Complex I) was most severely impaired in the mutant, whereas its activity was restored in a revertant of the mutant that had regained the ability to synthesize CL. These results suggest that CL plays a critical role in mitochondrial functions, at least in the respiratory electron transport chain.     

ERCC1 mutations in UV-sensitive Chinese hamster ovary (CHO) cell lines

Bronchodilatory substances such as the phosphodisterase inhibitor (PDE-I) theophylline stimulate mucociliary activity. With the introduction of selective PDE-Is it has become possible to study the functional importance of each phosphodiesterase enzyme (PDE) concerning the regulation of the ciliary beat. The effects of rolipram (inhibiting a cAMP specific PDE (PDE4), milrinone (inhibiting a cGMP inhibited PDE (PDE3)) and zaprinast (inhibiting a cGMP specific PDE (PDE5)) were investigated in in vitro preparations from the rabbit maxillary sinus and trachea. Ciliary beat frequency (CBF) was measured with a photoelectrical method. In sinus mucosa all three compounds accelerated CBF. Milrinone (10(-5) M) by 22.6 +/- 5.3% (n = 6; P < 0.01), rolipram (10(-5) M) by 29.7 +/- 5.7% (n = 7; P < 0.01), and zaprinast (10(-5) M) by 19.4 +/- 6.3% (n = 6; P < 0.05). In the tracheal specimens at a concentration of 10(-5) M, milrinone accelerated CBF by 27.5 +/- 9.0% (n = 7; < 0.05), rolipram by 11.6 +/- 2.8% (n = 6; P < 0.05) and zaprinast by 24.3 +/- 5.3% (n = 7; P < 0.01). Comparison of the effects in the upper and lower airways showed that at concentrations of 10(-5) and 10(-4) M rolipram was more effective in the upper than in the lower airways. The reverse was true of milrinone which concentrations of 10(-7) and 10(-6) M had a significant effect in tracheal specimens but not in sinus specimens. Zaprinast was equally effective in both the upper and lower airways. It is concluded that in both the upper and lower airways selective PDE-Is have an accelerating effect on the CBF that may be beneficial in the treatment of airway diseases.     

Proton and chloride currents in Chinese hamster ovary cells

Despite being one of the most frequent neoplasms occurring in the endocrine system, thyroid carcinoma is, nevertheless, a relatively rare event (0.5-1.5% of all malignant tumours in man); the differentiated forms are the most prevalent and are characterized by a high mean survival rate, whereas the very aggressive forms are rare and prognosis is unfavourable. Diagnostic evaluation of carcinomatous lesions, particularly in the early stages, may give rise to considerable difficulties at a clinical level due to the differentiation of the benign lesions, which are a frequent finding. The traditional clinico-semeiological and instrumental parameters, which, in the past, were used in the assessment of suspected malignancy, should not be considered as markers of malignancy; however, exposure to ionizing radiations during childhood may have a well defined role of risk. Following the recent progress in genetic and molecular studies, it is now possible to exploit genetic-molecular tumor markers and, at present, thyroid medullary carcinoma may be identified also in the absence of clinical evidence, particularly the familial form, thus allowing suitable prophylaxis in those subjects with specific genetic impairment (e.g. preventive thyroidectomy in infancy). Since no discriminating clinico-semeiological parameters are available, considering the aspecificity of scintigraphic findings and the lack of reliability of echographic imaging in providing data which enable us to distinguish a rare neoplastic pattern from the more frequent finding of a benign thyroid mass, fine-needle aspiration (FNA) cytology may today be considered the technique of choice in the screening of the thyroid nodule. Our experience in over 12,000 nodular lesions since 1982, has confirmed that the cytological examination is the most discriminating investigation, diagnostic reliability being far greater than that of traditional techniques. Considering the high frequency of thyroid nodule disease which rarely harbours a carcinomatous lesion, a very scrupulous diagnostic algorithm is mandatory. The FNA cytology, together with morphofunctional and immunological examinations, as well as dynamic exploration of the thyroid hypothalamo-pituitary axis, which allows a nosographic picture of the thyroid nodule disease, provides a more discriminating appraisal for the surgical approach to a single, solitary or prominent nodule.     

Preferential retention of the mink chromosome group in somatic cell hybrids of Chinese hamster and American mink

The chromosomal complement was studied in 57 independent clones of hybrid cells obtained in six experiments for fusion of Chinese hamster and American mink cells. Various mink chromosomes in hybrids of concern are lost. It is shown when a small number of mink chromosomes are retained in hybrid clones, chromosomes 6, 9, 10, 12, 13, 14 and X segregate in a similar way and are preferentially retained in these clones. When a considerable number of mink chromosomes are detained in hybrid clones, the segregation mode is close to a random one.     

Ammonia inhibits neural cell adhesion molecule polysialylation in Chinese hamster ovary and small cell lung cancer cells

Contrasts between implicit and explicit knowledge in the serial reaction time (SJRT) paradigm have been challenged because they have depended on a single dissociation; intact implicit knowledge in the absence of corresponding explicit knowledge. In the SRT task, subjects respond with a corresponding keypress to a cue that appears in one of four locations. The cue follows a repeating sequence of locations, and subjects can exhibit knowledge of the repeating sequence through increasingly rapid performance (an implicit test) or by being able to recognize the sequence (an explicit test). In our study, amnesic patients were given extensive SRT training. Their implicit and explicit test performance was compared to the performance of control subjects who memorized the training sequence. Compared with control subjects, amnesic patients exhibited superior performance on the implicit task and impaired performance on the explicit task. This crossover interaction suggests that implicit and explicit knowledge of the embedded sequence are separate and encapsulated and that they presumably depend on different brain systems.     

Selective isolation of reversible cold sensitive variants from Chinese hamster ovary cell cultures

The fine structure and cellular associations of the large pigment cells (LPC's) of the compound eye of the house fly were studied with high voltage and conventional electron microscopy. Depending on the sector of the compound eye, the facets are either rectangular or hexagonal. The underside of each facet has indentations exactly aligned with those on top into which inserts an angulated sleeve of LPC's. Under the rectangular lens facet 6 or 8 small compact (in cross section) LPC's join four elongate LPC's. Clusters of compact cells alternate in this ring with elongate ones. Compact cells compress together and become quadrangular (in cross section) several microns below their insertion into the lens and form "building block" corners while elongate cells form "side rails" for the rectangular type of distal pseudocone enclosure. Beneath hexagonal facets all LPC's are rather elongate with out corner cells. In both facet types LPC's enclose the pseudocone for a longitudinal distance of 4 mum and then are displaced as bordering cells by a sleeve of two corneal pigment cells (CPC's), each of which encloses half of the proximal pseudocone. For the following 6 mum of longitudinal distance these concentric sleeves of CPC's and LPC's form a double layer around the pseudocone. At about 10 mum below lens base the two sleeves separate; LPC's become attenuated and extend cable-like to the basement membrane and CPC's enclose the proximal pseudocone, Semper cells and distal retinula. The junction between lens and LPC's has critical structural value in that (1) this is the sole anchorage to the lens by the lengthy remainder of the ommatidium, and (2) LPC's enclose the semiliquid pseudocone in the most distal portion of the pseudocone. In addition to vertical support, the LPC's send out numerous lateral processes that make structural contact among themselves, with the corneal pigment cells and the photoreceptor cells. The structural features of this array are discussed relative to possible physiological roles.     

Apoptosis in batch cultures of Chinese hamster ovary cells

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum-free batch culture of CHO cells in suspension died via apoptosis--as evidenced by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases of the Interleukin-1-beta-Converting Enzyme (ICE) family, e.g., CPP32, Mch-1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoro-methyl-ketone (z-VAD.fmk), was unable to substantially extend the life of a serum-free batch culture of CHO cells. In addition, z-VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival factors, insulin and transferrin. In both these instances, z-VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl-2 expression was able to significantly extend viabilities in CHO batch culture. Bcl-2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell.     

Effect of cytolethal distending toxin on F-actin assembly and cell division in Chinese hamster ovary cells

Cytolethal distending toxin (CDT) is a newly described toxin produced by a number of enteropathogens, including Campylobacter jejuni, various Escherichia coli strains, and a few Shigella species. CDT induces distension and eventual death of a number of transformed cell lines. Here, we extend previous studies by demonstrating that morphological changes in CDT-treated Chinese hamster ovary cells are coincident with changes in cytoskeletal structure and an inhibition of cell proliferation. CDT-treated cells underwent a progressive accumulation of F-actin assemblies which microscopically resembled actin stress fibers. Accumulation of the stress fiber-like structures in CDT-treated cells was accompanied by an apparent blockage of cell division. Multinucleation was detected in some cells but did not constitute a significant feature of CDT action. Although toxin-treated cells failed to divide, cell viability remained high for the first 4 days following toxin treatment, as evidenced by trypan blue exclusion and neutral red uptake. [3H]thymidine incorporation studies on CDT-treated cells were consistent with a blockage of cell proliferation without a direct inhibition of DNA synthesis. Although the progression of toxin action developed slowly, a 2-min exposure to CDT resulted in an irreversible development of toxicity. Together, our data indicate that CDT affects F-actin assembly within target cells and may interrupt the regulation or function of cell cycle-dependent events leading to cytokinesis.     

Production of recombinant proteins in Chinese hamster ovary cells using a protein-free cell culture medium

The growth-factor prototrophic Chinese hamster ovary (CHO) SSF3 cell line was previously adapted for growth in serum-free media. Here we present a newly designed medium which allows these cells to grow in the absence of any exogenously added growth factors. To investigate the capacity of CHO SSF3 cells for the efficient production of recombinant proteins in protein-free media, expression plasmids containing either a human single chain urokinase-type plasminogen activator (uPA)-encoding cDNA or a humanized immunoglobulin G (IgG) kappa light chain cDNA were introduced by transfection. The tryptophan synthase (trpB) gene of Escherichia coli was used as a dominantly acting selection marker allowing the cells to survive in a medium containing indole in place of tryptophan. Some of the clones obtained exhibited a stable uPA expression over a period of several months under selective conditions and the yields were up to 74 mg of uPA/l in a bioreactor and the productivity was around 40 mg/day per 10(9) cells. The yields of IgG light chains were up to 118 mg/l and the productivity was in the order of 56 mg/day per 10(9) cells in a bioreactor. These results demonstrate the potential of CHO SSF3 cells for the efficient production of recombinant proteins under protein-free conditions.     

Using mitochondrial and nuclear DNA markers to reconstruct human evolution

Molecular genetic-data have greatly improved our ability to test hypotheses about human evolution. During the past decade, a large amount of nuclear and mitochondrial data have been collected from diverse human populations. Taken together, these data indicate that modern humans are a relatively young species. African populations show the largest amount of genetic diversity, and they are the most genetically divergent population. Modern human populations expanded in size first on the African continent. These findings support a recent African origin of modern humans, but this conclusion should be tempered by the possible effects of factors such as gene flow, population size differences, and natural selection.     

Modulation of membrane drug permeability in Chinese hamster ovary cells

The kinetics of colchicine uptake into Chinese hamster ovary cells have been investigated and found to be consistent with an unmediated diffusion mode. A variety of compounds such as local anesthetics and non-ionic detergents as well as drugs such as vinblastine, vincristine, daunomycin and actinomycin D potentiate the rate of colchicine uptake into these cells and into colchicine resistant mutants. In all cases, higher concentrations of these compounds were required to stimulate colchicine uptake in the colchicine resistant mutants than in the cells of the parental line. This stimulation was observed also in the uptake of puromycin, a structurally and functionally different drug. These stimulatory agents did not, however, cause the cells to become nonspecifically leaky since the uptake of 2-deoxy-D-glucose was unaffected. In addition, the activation energy of colchicine uptake was unaltered in the presence of stimulating agents, implying that they were not causing colchicine to enter the cells via a different mechanism. The results are compatible with the view that these compounds are membrane-active, and are able to stimulate an increased rate of unmediated diffusion of colchicine into the cells. It appears that a mechanism for the regulation of passive permeability is modified in the resistant mutants.     

Ordered cell cycle phase perturbations in Chinese hamster ovary cells treated with an S-adenosylmethionine decarboxylase inhibitor

The polyamines putrescine, spermidine, and spermine are needed for normal cell cycle progression and polyamine-depleted cells cease to proliferate. We have investigated cell cycle perturbations in Chinese hamster ovary cells seeded in the presence of 4-amidinoindan-1-one 2'-amidinohydrazone (CGP 48664), a potent inhibitor of S-adenosylmethionine decarboxylase, an enzyme which is essential for the synthesis of spermidine and spermine. At 9 h and at 1, 2, and 3 days after seeding, cells were labelled with the thymidine analog bromodeoxyuridine (BrdUrd) for 30 min, after which the BrdUrd-containing medium was removed and the cells were allowed to progress through the cell cycle in BrdUrd-free medium before sampling (post-labelling time). Using flow cytometry, coupled with an indirect immunofluorenscence technique, utilizing monoclonal anti-BrdUrd and secondary fluorescein-isothiocyanate-conjugated antibodies, and the DNA stain propidium iodide, cellular BrdUrd and DNA contents were quantified. By investigating the movement of BrdUrd-nonlabelled G1 cells into S phase during the post-labelling time, a measure of the G1/S transition was obtained. The time of appearence in G1 of BrdUrd-labelled cells which had divided was used to monitor the length of the G2+M phase. A measure of the S phase length (DNA synthesis time) was obtained by monitoring the progression of BrdUrd-labelled cells through S phase. CGP 48664-induced spermine depletion significantly increased the length of the S phase already 9 h after seeding cells in the presence of the inhibitor. No effects on the G1/S transition or on the length of the G2+M phase were observed until 2 days after seeding.     

Overexpression of hormone-sensitive lipase in Chinese hamster ovary cells leads to abnormalities in cholesterol homeostasis

Hormone-sensitive lipase (HSL) is an intracellular enzyme that functions as both a neutral triglyceride and cholesteryl ester hydrolase. In order to explore the effects of HSL on cholesterol homeostasis, Chinese hamster ovary (CHO) cells were transfected with rat HSL and several different stable cell lines that overexpress HSL mRNA, HSL protein, and HSL activity approximately 600-fold were isolated. Cells transfected with HSL contained less cholesteryl esters and unesterified cholesterol than control cells. HSL transfectants expressed 20-60% fewer LDL receptors than control cells when grown in lipid-depleted media or in the presence of mevinolin, as assessed by binding and degradation of LDL and immunoblotting of LDL receptors. In contrast, the rate of cholesterol synthesis and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were increased 3- to 14-fold in HSL transfectants grown in sterol replete media. The rate of cholesterol synthesis and the activity of HMG-CoA reductase increased when cells were grown in lipid-depleted media, and remained markedly elevated compared to control cells. These results show that the regulation of LDL receptor expression and cholesterol synthesis can be dissociated through the actions of HSL and suggest multiple control mechanisms for sterol-responsive genes.