Cloning Efficiency Of Cells Research Articles

Effect of leukocytic serum preparation on hematopoietic cells in vitro

The effects of leucocyte serum (LS) on bone marrow cells (BMC), thymus and HL-60 human myeloid leukemia cells were studied in liquid suspension and agar cultures. LS increased 3H-thymidine incorporation in BMC and intensified the cloning efficiency of granulocyte-macrophage progenitor cells (CFU-GM) and human myeloid leukemia cells. No significant stimulatory effect on thymus cells was observed. It has been shown that LS prevents or markedly decreases the effect of granulocyte inhibitor (GI-3S2).

83] Measurement of effect of interferons on cloning efficiency of primary tumor cells in culture

This chapter discusses the measurement of the effect of interferons on the cloning efficiency of primary tumor cells in culture. The biological properties initially associated with interferons are their antiviral activities, but they are subsequently shown to have antiproliferative effects against a variety of cell types, both in vitro and in vivo . To evaluate the antiproliferative activity of natural and recombinant human interferons in a model that might prove predictive of clinical antitumor response, the human tumor clonogenic assay (HTCA) is utilized on fresh biopsies obtained from cancer patients. HTCA has previously been applied to chemosensitivity testing with standard cytotoxic anticancer drugs and found to be predictive of clinical response in cancer patients. The assay has been applied to in vitro phase II testing of a variety of new anticancer drugs. The results of in vitro studies suggest that both natural and recombinant interferons tested have antitumor activity against a variety of human cancers but may differ somewhat in their potency and specificity for tumors of differing histologic origin.

In vitro DNA strand scission and inhibition of nucleic acid synthesis in L1210 leukemia cells by a new class of DNA complexers, the anthra[1,9- cd]pyrazol-6(2 H)-ones (anthrapyrazoles)

CI-937 and CI-942 belong to a new class of DNA complexes, the anthra[1,9- cd]pyrazol-6(2 H)-ones (anthrapyrazoles), and are being further developed as antitumor drugs based on their curative properties against murine solid tumour models. The biochemical effects of these agents were studied in L1210 leukemia in relation to other clinically used intercalators. After a 1-hr exposure, CI-937 and CI-942 reduced the cloning efficiency of L1210 cells by 50% at 3.0 × 10 −8 and 1.5 × 10 −7 respectively. Based on an ethidium displacement assay, these drugs bound strongly to DNA, reducing the fluorescence of an ethidium-DNA complex by 50% at concentrations of 23 and 33 nM for CI-937 and CI-942 respectively. This was comparable to mitoxantrone at 15 nM, but much more potent than Amsacrine which required over 1.3 μM. A distinct property of the anthrapyrazoles was a much more potent inhibitory effect on whole cell DNA synthesis than on RNA synthesis. After L1210 cells were exposed to drug for 2 hr the concentration needed to inhibit DNA synthesis by 50% was 0.33 and 0.57 μM for CI-937 and CI-942, respectively, whereas 2.0 and 11.3 μM were required to inhibit RNA synthesis by the same extent. This was in contrast to Adriamycin and mitoxantrone which inhibited both activities equally at similar concentrations. It was apparent that the inhibition of these processes was not due to substrate depletion since intracellular ribonucleoside and deoxyribonucleoside triphosphates either remained constant or were elevated after a 2-hr exposure to 1 or 10 μM drug. A similar discriminatory effect was observed on DNA and RNA polymerase in permeabilized cells, and the inhibition of nucleic acid synthesis in this system could be reversed by exogenously added DNA. Since the high incidence of cardiotoxicity associated with the administration of anthracyclines has been related to the formation of reactive oxygen species, the ability of the anthrapyrazoles to augment superoxide dismutase sensitive oxygen consumption was observed in a rat liver microsomal system. CI-937 and CI-942 induced 5- and 10-fold less oxygen consumption than Adriamycin, producing rates of 12.4, 24.2 and 138.9 nmoles/min/mg microsomal protein, respectively, at a drug concentration of 0.5 mM. The anthrapyrazoles were similar to other known intercalators in that they caused both single- and, to a lesser extent, double-strand DNA breaks that were tightly associated with protein in a concentration-dependent manner. The rates at which these lesions formed, however, were quite different between drugs. CI-937 at a concentration of 1 μM produced maximum strand breakage after 30 min, whereas CI-942 was much slower requiring at least 3 hr. If L1210 cells were treated with the anthrapyrazoles such that substantial strand breaks occurred and the drug removed, the breaks were repaired very slowly over the first 30 min but then additional lesions occurred thereafter for at least 2 hr. Additional DNA damage after drug was removed was also observed with Adriamycin and mitoxantrone but not Amsacrine with which 90% of the strand breaks disappeared within 30 min.

Rapid fluorometric detection of drug resistant tumour cells.

A rapid estimation of mutation frequency in tumours would be invaluable in cancer management. Considerable evidence suggests that drug resistance in tumours frequently arises as a consequence of spontaneous somatic mutation (Goldie & Coldman, 1979) and therefore the mutation rate of a tumour will be an index of the potential to develop drug resistance. Previous studies have compared the mutation rates of cell lines using cloning assays. Cifone & Fidler (1981) reported a higher mutation rate in metastatic variant of UV-2237 fibrosarcoma cells than in a clone of the same cell line with lower metastatic potential. Warren et al. (1981) reported that fibroblasts from patients with Bloom syndrome, which predisposes individuals to various cancers, had a 10-15 fold higher mutation rate than did fibroblasts from normal individuals. These findings have been important in correlating malignant capacity with genetic instability, but unfortunately, the techniques used have limited general application since few human tumour cells will form colonies on plastic. An alternative assay, soft agar cloning, has also been used for the determination of mutation rates in a variety of mammalian cell lines, including Chinese hamster ovary cells (Li & Shimizu, 1983) and the L5178Y mouse lymphoma cell line (Irr & Snee, 1982). Again, the low cloning efficiency of human tumour cells in soft agar (Hamburger et al., 1978) and the fact that this method selects only mutant cells which clone in agar limits the use of this assay to determine mutation frequency in human tumours. This latter source of error might bias the estimation of the mutation rate in favour of more malignant cells, which generally have higher cloning efficiencies (Elmore et al., 1983). Morley et al. (1983) and Albertini et al. (1982) have reported a limiting dilution technique for the measurement of mutation frequency in human lymphocytes. Culture conditions have been optimised for peripheral blood lymphocytes and a cloning efficiency of 20-60% has been obtained. Problems have been encountered, however, in studies of cultured lymphoblast lines with variable

In vitro culture of leukemic cells in t(4;11) acute leukemia

In the present study we utilized a semisolid culture system with feeder cells and enriched media to evaluate the growth of acute leukemia associated with the 4;11 chromosomal translocation. We compared growth of t(4;11) leukemia to typical acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). The two cases of t(4;11) leukemia tested exhibited the highest cloning efficiency of cells tested. The growth characteristics of t(4;11) leukemia were more similar to ANL than ALL.

In Vitro Culture of Leukemic Cells in t(4;11) Acute Leukemia

In the present study we utilized a semisolid culture system with feeder cells and enriched media to evaluate the growth of acute leukemia associated with the 4;11 chromosomal translocation. We compared growth of t(4;11) leukemia to typical acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). The two cases of t(4;11) leukemia tested exhibited the highest cloning efficiency of cells tested. The growth characteristics of t(4;11) leukemia were more similar to ANL than ALL.

Enhanced Cloning Efficiency of Murine Rhabdomyosarcoma Cells After Chlorozotocin Treatment: Relationship With Enhanced Lung Metastasis

The effect of chlorozotocin [(CZT) CAS: 54749-90-5; 2-(3-(2-chloroethyl)-3-nitrosoureido)-D-gluco-pyranose] was studied on a series of tumor cells, cultured or extracted fresh primary or transplanted tumors, by means of clonogenic assay. The ability of most rat rhabdomyosarcoma cells to form colonies in soft agar was enhanced when exposed to the water-soluble nitrosourea chloride CZT. The tumor cells tested were derived from a) several primary tumors induced in WAG rats by colloidal nickel, then cultured and exposed to CZT early during in vitro passage; b) the 9-4 tumor, also Ni-induced but maintained in long-term culture; and c) the Ni-induced 9-4/0 tumor, maintained by transplantation in syngeneic rats. No inhibition of colony formation was observed in any of the cell lines even at high concentrations of CZT. Adriamycin, chosen as a control treatment, strongly inhibited the cloning efficiency (CE) of the tumor cells. In vivo, the weekly injection of 10 mg CZT/kg body weight into syngeneic rats bearing transplanted tumors led to an enhancement of lung metastasis formation. The CZT enhancement of CE of tumor cells and its relationship to increased in vivo tumor metastasis is discussed.

Role of retinoids in differentiation and growth of embryonal carcinoma cells.

To study how retinoids promote differentiation and inhibit proliferation of embryonal carcinoma (EC) cells, we have followed their intracellular fate. Retinoic acid (RA) is effectively metabolized to more polar compounds by many EC lines. Unlike RA, retinol is slowly metabolized. Our inability to detect conversion of retinol to RA might indicate that the two retinoids elicit their effects on EC cells in different ways. Retinol added to cultures quickly appears in the nuclear fraction; the proportion associated with nuclei after detergent extraction is initially very low but increases with time. Retinol and RA might be translocated to nuclei by their respective binding proteins [cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP)]: isolated EC nuclei have specific, independent binding sites for both holoproteins but not their ligands. CRABP cannot be detected in the nucleoplasm of untreated EC cells, but activity is measurable after cells are exposed to RA. Interestingly, incubation with retinol promotes movement of both CRBP and CRABP into the nucleoplasmic fraction. Finally, we have demonstrated that brief exposure to RA dramatically reduces the cloning efficiency of EC cells. Since some cells are unaffected even by lengthy exposures to RA whereas the growth of their progeny is inhibited, we suggest that EC cells can become epigenetically refractory to RA.

Cloning Efficiency of Cultured Human Tumor Cell Lines Measured With the Use of a Coulter Particle Counter<xref ref-type="fn" rid="FN2">2</xref>

A technique is described by which colony formation in agarose may be rapidly and reproducibly determined with the use of a modified Coulter particle counter (CPC). The cloning efficiency of RD human rhabdomyosarcoma cells after exposure to vincristine sulfate or cisplatin has been compared with the CPC method or by conventional visual counting. These techniques give very similar results. In combination with a Coulter Channelyzer, the CPC technique can be used to evaluate drug effects in colony sizes from a median of 20 to greater than 290 cells. The integrity of colonies formed by 3 colon carcinoma cell lines was examined to determine if this method could be applied to other systems.

The toxicity of sixteen metallic compounds in Chinese hamster ovary cells

The toxicity of 16 metal salts to Chinese hamster ovary (CHO) cells was determined by measuring the cloning efficiency (CE) of CHO cells after exposure to the metals. CHO cells differed by a factor of 10 5 to 10 6 in their toxic response to these metal salts. While Cd(II) was the most toxic ion, Mg(II) exhibited the least toxicity based on either CE50 (concentration required to reduce the CE to 50%) or D 0 (concentration increment which reduced the CE by 63%). On the basis of CE50, the toxicity ranking was Ag > Tl for monovalent metals, Cd > Zn > Hg > Co > Cu > Mn > Ni > Be > Pd > Sr > Mg for divalent metals, and In > Rh > Y for trivalent metals. A similar ranking was found for D 0. For the 11 divalent metals, correlations of CE50 and D 0 in the CHO cell assay and the Pearson-Mawby softness parameter for metals (δ p) were reasonably strong. A good correlation exists between the results of this study on the toxic response in CHO cells and published data on toxicity in mice and Drosophila. It appears that the CHO cell cloning assay may be useful in preliminary screening of metallic compounds as an indicator of their predicted toxicity in higher organisms.

Potential biological explanation of stimulation of colony growth in semi-solid agar by cytotoxic agents.

Br. J. Cancer (1983), 48, 863-868 Short Communication Potential biological explanation of stimulation of colony growth in semi-solid agar by cytotoxic agents F.L. Meyskens, Jr., S.P. Thomson, R.A. Hickiel & N.J. Sipes Cancer Center Division and Department of Internal Medicine, University of Arizona, Tucson, Arizona 85724, USA and 'Department of Pharmacology, University of Saskatchewan, Saskatoon. Canada S7N OWO. Growth of single cells into colonies in semisolid medium has been widely used both as a measure of the clonogenic potential of normal and transformed cells (Park et al., 1971; Thomson & Rauth, 1974; Courtenay, 1976; Metcalf, 1977; Buick et al., 1977; Hamburger et al., 1977; Salmon, 1980) and as an in vitro marker for cellular transformation (Macpherson & Montagnier, 1964; MacAllister & Reed, 1968). Growth of cells in semisolid medium also has been used to measure the effect of various cytotoxic and non-cytotoxic agents on clonogenic tumour cells (Salmon et al., 1978; Tveit et al., 1980, 1982; Von Hoff et al., 1981; Meyskens et al., 1981). The relation between the number of cells plated and the formation of clusters and colonies, defined here as growth units, should be clearly defined to assure valid interpretation of perturbations of clonogenic cells. The frequency and extent of proliferation to form growth units has been regarded as a function of the clonogenic capacity of the tumour sample together with the conditions of culture. However, many of the effects of the conditions of culture remain undefined. Therefore, because clonogenic assays are generally closed non re-fed systems, we have examined the relationship between the number of cells plated, the number of growth units formed, the relative frequency of growth units containing different numbers of cells, and the total number of cells formed within the growth units. We found that the cloning efficiency and proliferative characteristics of clonogenic cells in agar is significantly determined by the number of cells plated. Murine melanoma cells (CCL 53.1) were grown in medium (FIO plus 10% horse and 2% heat- inactivated foetal calf-serum) as monolayers in plastic Falcon flasks. Treatment with Tryodes solution removed the cells and produced suspensions of single cells. Different numbers of cells were plated in 1.0ml of medium containing Correspondence: F.L. Meyskens. Received 10 May 1983; accepted 6 September 1983. 0.3% agar (Bacto) over 1.0ml of 0.5% agar in medium in 35mm diameter Petri dishes. The plates were incubated in a humidified, 5% Co2, air atmosphere at 37°C for 18 days. Every group of cells (containing greater than one cell) was counted in randomly selected 1, 2.5, or 6.25 mm2 areas. The mean +s.e. number of growth units per area was multiplied by 908 mm2 per 35mm culture dish to obtain the total number of growth units per culture dish. The number of growth units of different diameters was calculated using the relative frequency of growth units by size, obtained by direct measurement of 200 growth units per plate with a micrometer, and multiplying by the total number of growth units per plate. A nomogram was constructed to determine the number of cells per growth unit. This was done by direct visual observation in growth units containing 80pm in diameter. As the number of cells plated was decreased, the number of larger growth units increased. For example, when 500 cells were plated, only 10% of colonies were The Macmillan Press Ltd., 1983

Selective killing of target cells by antibody-ricin A chain or antibody-gelonin hybrid molecules: comparison of cytotoxic potency and use in immunoselection procedures.

Ricin A chain and gelonin, two plant proteins that can powerfully reduce the protein synthetic capacity of ribosome preparations, were covalently coupled to a monoclonal anti-Thy-1.2 antibody. Two conjugates were prepared by using N-succinimidyl-3-2(-pyridyldithio)propionate, which generates a disulfide linkage between the component molecules. Both conjugates specifically killed Thy-1.2 antigen-bearing EL-4 cells, but had no effect on Thy-1.2- BW5147 cells. The cytotoxic potency of both reagents was determined by comparing the cloning efficiency of E1-4 target cells after treatment with the conjugate. The frequency of cells surviving treatment with 45 micrograms/10(6) cells of the gelonin conjugate was 1/72, whereas this frequency was 1/836 after exposure to 7 micrograms of the Ricin A chain conjugate. Both reagents could be successfully used to select Thy-1.2- cells from cell mixtures consisting of Thy-1.2+ and Thy-1.2- cells.

Differential Growth Regulation of Human Bladder Tumor Cells (J82) in Culture by Normal Bladder Cells from Different Aged Human Donors

This study used an agarose gel culture system to study growth regulatory interactions between normal and neoplastic human bladder cells. The effects of normal human fibroblasts on tumor cell cloning efficiency in agarose was studied using 2 experimental protocols. In 1 system, the fibroblasts proliferated as anchored cells beneath the agarose, and in the other the fibroblasts were nongrowing cells embedded within the agarose as nonanchored cells. In contrast to the largely inhibitory effects of anchored fetal fibroblasts on tumor cell growth, their nonanchored counterparts were exclusively stimulatory. Similar experiments with normal fibroblasts derived from the bladders of increasingly older donors showed a progressive decline in their capacity to inhibit tumor cell growth in agarose. Further experiments modulating the growth rate of neonatal fibroblasts with fetal bovine serum have indicated that the diverse tumor responses elicited by anchored versus nonanchored cells, as well as those observed for the aged bladder fibroblasts, were a function of fibroblast growth rate. We concluded that there was an inverse regulatory relationship between the growth of normal and tumor cells; rapidly growing fibroblasts were inhibitory whereas nongrowing fibroblasts stimulated tumor cell growth in agarose culture.

Single cell studies on hapten-specific B lymphocytes: differential cloning efficiency of cells of various sizes.

Three separate forms of in vitro stimulation were assessed for their capacity to activate hapten-gelatin fractionated, fluorescein- (Flu) specific murine splenic B lymphocytes. They were: a) Flu-polymerized flagellin (Flu-POL) acting on a single Flu-specific B cell in microculture in the absence of "filler" or feeder cells, but in the presence of T cell-derived B cell growth and differentiation factor(s) (BGDF); b) a mixture of mitogens, E. coli lipopolysaccharide (LPS) and dextran sulfate, acting on a single Flu-specific B cell in the absence of added BGDF; and c) Flu-POL plus BGDF acting on single Flu-specific B cells as in a but with thymus filler cells also present. System c was markedly superior in causing antibody formation, 15 to 22% of cells forming a clone of Flu-specific antibody-forming cells (AFC), in contrast to 6% for system b and 3 to 6% for system a. Each stimulus was applied to single cells that had been size fractionated into samples of increasing size by using the forward light-scattering parameter of the fluorescence-activated cell sorter. Surprisingly, the smaller sized fractions proliferated poorly in system a and contributed less than 10% of the antibody-forming potential of the total population. The smaller cells proliferated better in system b, but only 10 to 15% of proliferating clones generated Flu-specific AFC, whereas the larger cells contributed 86% of the total AFC response. Even in system c, only 6% of the small cells formed AFC clones compared with 41% of the larger cells. It thus appears that the smaller half of murine B lymphocytes is relatively resistant to activation into proliferation and differentiation by "T-independent" antigens; when activated by mitogens, they clone less efficiently than larger cells. Despite these limitations, system c could generate a total of up to four hapten-specific AFC for every B cell placed into culture, making it the most efficient system of specific antibody formation yet described.

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, 2- acetamido-1,3,4,6- tetra-O- acetyl-2- deoxy- d-mannopyranose (Ac 4-NAcMan) and the 2-trifluoroacetamido derivative (Ac 4F 3-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of N-[ 3 H]acetylmannosamine into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac 4F 3-NAcMan and Ac 4-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac 4-NAcMan was relatively reversible, whereas that produced by Ac 4F 3-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of N-[ 3 H]acetylmannosamine into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac 4F 3-NAcMan caused accumulation of radioactivity from N-[ 3 H]acetylmannosamine in CMP- N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound 3H caused by this agent. In contrast, similar exposure to Ac 4-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from N-[ 3 H]acetylmannosamine in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with 3H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac 4-NAcMan labeled with 14C in the 4- O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.

Clonal analysis of insulin and somatostatin secretion and L-dopa decarboxylase expression by a rat islet cell tumor.

A cell line, RIN-m, established from a transplantable rat islet cell tumor secretes insulin (IRI) and somatostatin (SRIF) and expresses high levels of the key amine precursor uptake and decarboxylation (APUD) cell enzyme L-dopa-decarboxylase (DDC). Conditioned medium from a rat pituitary tumor line GH3, secreting GH and PRL, improved the cloning efficiency of RIN-m cells 24-fold and enabled the isolation and establishment of a large number of primary and secondary clones. These clones were used to study clonal relationships between peptide hormone secretion and APUD features of an endocrine cell. All the primary and secondary clonal derivatives, irrespective of whether they secreted peptide hormones, maintained high levels of DDC activity. In contrast, IRI and SRIF secretion patterns of the primary clones were highly variable. Selective recloning of primary clones resulted in the isolation of subclones which produced either no hormones or high levels of either IRI or SRIF, but no clone that continuously secreted high levels of both IRI and SRIF. We conclude that: 1) the rat pituitary tumor line GH3 produces a factor(s), possibly GH and/or PRL, which dramatically affects the growth and cloning efficiency of rat islet tumor cells; 2) in contrast to the variability in hormone secretion patterns, DDC activity was consistently expressed in all clones and subclones; and 3) although wide fluctuation in hormone secretion levels occurred among the primary clones, subclones were obtained which revealed that IRI and SRIF can be expressed independently. The subclones of RIN-m developed should be useful for the analyses of factors influencing the synthesis, storage, and secretion of IRI and SRIF. The persistence of high DDC activity in the primary and secondary clones suggests that the APUD property of this endocrine cell may be a primitive differentiation feature closely related to the stem cell; in contrast, peptide hormone production may be associated with more terminal differentiation events.

Cell detachment and cloning efficiency as parameters for cytotoxicity

Cell detachment and cloning efficiency of Baby Hamster Kidney cells (BHK-21 C13) were used as parameters to quantify cytotoxicity in vitro of 3 endogenous chemicals (glutathione, L-methionine, L-cysteine HCl) and 4 organotin compounds (tributyltinoxide, tributylchloride, tetrabutyltin, tetraphenyltin). IC 50 values (inhibitory concentration at which the cloning efficiency was reduced to 50%) were estimated to be larger than 10 −3 M for all 3 endogenous substances, which served as a calibration of the cell culture system for non-toxic chemicals. For the 2 tributyltin salts the IC 50 values were estimated to be near 10 −6 M and for the 2 tetraalkyltin compounds near 10 −5 M. The estimated CD 50 values (concentration at whicb 50% of the cells detach) were at least twice as large as the corresponding IC 50 values for all 7 chemicals tested. For the toxic tributyltin salts the cell detachment assay was 30–60 times less sensitive than the cloning efficiency assay. However, both assays rank all compounds tested in the same sequence of toxicity as that known from in vivo studies.

Interaction between latent papovavirus genomes and the tumor promoter TPA

The tumor promoting agent 12-O-tetradecanoylphorbol-13acetate (TPA) enhances both the transformation of rat embryo cells by adenovirus and the cloning efficiency of such cells [ 11. Induction of Epstein-Barr virus (EBV) antigens and stimulation of particle synthesis by treatment of the EBV-carrying cell lines with TPA was reported in [ 2,3]. Here we report that TPA exclusively enhances the viral DNA content of cells which harbor papovavirus genomes in a latent episomal state. On the other hand, no effect is exerted by TPA on the copy number of the viral DNA in productively infected cells and on integrated papovavirus DNA in transformed cells.

Clonal agar culture of normal primary explanted bovine granulosa cells

When seeded at low cell densities (500 to 2500 cells per dish), normal primary explanted bovine granulosa cells grow in a double layer semi-solid agar culture system as anchorage-independent clones composed of well differentiated cells with vacuolated granular cytoplasm with a cloning efficiency of between 2% and 4%. The addition of growth promoting factors to the culture medium such as epidermal growth factor (EGF), fibroblast growth factor (FGF) or rat erythrocyte suspensions resulted in a 5 to 6-fold increase in colony numbers and this was accompanied by an increase in colony size. Cells from both small (3–7 mm diameter) and large (>1 cm diameter) ovarian follicles exhibited similar patterns of responsiveness to growth factors but the cloning efficiency of cells from large follicles was approximately double that of cells from small follicles.

Dependence on stage of growth of mammalian cell sensitivity to near-UV irradiation.

Abstract The variable sensitivity of exponentially growing mouse myeloma cells (66.2 subclone of MPC11) to monochromatic near‐UV (365 nm) radiation was studied by determination of the relative cloning efficiency (i.e. survival) of cells in soft agar. Maximum sensitivity of the cells to near‐UV light was found during the middle and late stages of exponential growth when the survival was only one‐tenth of that during the early stage of growth. In addition, the shapes of near‐UV survival curves changed with the stage of the cells irradiated. These data indicate that stages of growth can substantially alter the response of mammalian cells to near‐UV radiation. These data further suggest the importance of testing the response of the cells throughout the growth employed in the experimental procedure.