Thymidine kinase-deficient mutants ofaedes albopictus mosquito cells

Summary Aedes albopictus mosquito cells resistant to the thymidine analog 5-bromodeoxyuridine (BrdU) were obtained using a single-step selection procedure. The resistant cells were characterized with respect to growth in the presence of BrdU, incorporation of [³H]uridine and [³H]thymidine, and thymidine kinase activity in crude extracts. The LC₅₀ for TK-6 cells was 95μg BrdU/ml, and for TK-8 cells was 45μg/ml. Both clones incorporated [³H]uridine at levels corresponding to those in wild-type cells. TK-6 and TK-8 cells did not incorporate [³H]thymidine into acid-precipitable material, nor did they contain measurable thymidine kinase activity. Thymidine kinase activity in crude extracts from wild-type cells had a Km of 2μM and a Vmax of 10 pmol · min⁻¹ · mg⁻¹ protein.     

Relationship between thymidine transport and phosphorylation in Novikoff rat hepatoma cells as analyzed by a rapid sampling technique

Incorporation of thymidine into Novikoff rat hepatoma cells was analyzed with a rapid sampling technique which allowed collection of 12 time points in 20 sec. Transport was studied in the absence of metabolism by using either ATP-depleted cells or a thymidine kinase negative subline. Transport was a rapid, saturable, nonconcentrative process with a K_m of about 85 μM. The intracellular thymidine pool was also rapidly labeled in cells which phosphorylated thymidine, so that a group translocation process involving thymidine kinase can be ruled out. Under all conditions examined, phosphorylation, not the transport, of thymidine was the rate-determining step in its incorporation into the acid-soluble pool. Estimation of transport rates from total incorporation into cells which phosphorylate the substrate is invalid in this cell system and must be questioned in all instances.     

Banded karyotypes of H-4-IIE-C3 rat hepatoma cells grown in vitro

Summary Mitotic cells from the H-4-IIE-C3 rat hepatoma tissue culture line showed a range of 45 to 53 chromosomes per cell with 75% of the cells displaying a chromosome number between 49 and 52. Analysis of Wright’s-Giemsa banded karyotypes of 22 cells revealed considerable cell to cell variation. Twenty-one structurally abnormal chromosomes were identified in these cells; the origin of nine of the 21 chromosomes were identified in these cells; the origin of nine of the 21 chromosomes could be determined. Of the structurally abnormal chromosomes detected, only one (M-1) occurred with a sufficiently high frequency to be of general use as a marker for these cells. This marker appears to be a Robertsonian translocation involving chromosome number 2 and chromosome number 10. This work was supported by grants-in-aid made available through the San Diego State University Foundation.     

Equilibrium binding of Hoechst 33258 and Hoechst 33342 fluorochromes with rat colorectal cells

We examined the biophysical characteristics of the interaction of Hoechst 33258 and 33342 dyes with normal rat colorectal cells as functions of fixation and solution composition. Classical dye-binding techniques were used to investigate the stoichiometry and binding constants with whole cells, and quantitative fluorescence image analysis was used to specifically study nuclear dye binding in intact cells. In aqueous solution, H-33258 dye bound cooperatively with intact cells, with a binding constant of between 3-4 x 10(5). In ethanolic solution, binding appeared less cooperative, although Scatchard analysis could not be used. The binding constant was slightly lower (2 x 10(5)), but the total number of cell binding sites was decreased by a factor of 5, reflecting a great decrease in cytoplasmic sites. QFIA studies identified conditions optimal for DNA quantitation under which the fluorescence signal was independent of dye or cell concentration. The proportionality between absolute nuclear fluorescence intensity and DNA content was established, and the upper limit of DNA content of normal colorectal cells was also determined.     

Control of angiotensinogen production by H4 rat hepatoma cells in serum-free culture

Summary A serum-free, hormone and attachment factor supplemented culture for rat H4 hepatoma cells was established. In the defined medium (Dulbecco's Modified Eagle's +Ham's F12+insulin, transferrin, fibronectin liver cell growth factor, and sodium selenite), H4 cells grew equally well as in 10% fetal bovine serum supplemented medium. H4 cells in either defined or serum-containing culture conditions produce transferrin but not albumin or alpha-fetoprotein. In this paper we have studied the effect of various hormones and pressor peptides on the production of angiotensinogen by H4 cells cultured in defined conditions. Only glucocorticoid hormone had a significant effect on the production of angiotensinogen, whereas other hormones previously reported to exert their effect on angiotensinogen production had little or no effect. This work was supported by grant P01 CA37589 from the National Institutes of Health, Bethesda, MD.     

Defective transport of thymidine by cultured cells resistant to 5-bromodeoxyuridine

A line of HeLa cells resistant to 5-bromo-2′-deoxyuridine (BUdR) was established by continuous culture in growth medium containing BUdR; during the selection period, BUdR concentrations, initially 15 μM, were gradually increased to 100 μM. Cells of a clone (HeLa/B5) established from this line were also resistant to 5-fluoro-2′-deoxyuridine (FUdR), but not to the free base, 5-fluorouracil. Although extracts of HeLa/B5 cells exhibited levels of thymidine kinase activity comparable to those of parental cells, rates of uptake of BUdR, FUdR, and thymidine into intact cells were much reduced. The kinetics of uptake of uridine and adenosine, nucleosides which appear to be transported independently of thymidine in HeLa cells, were similar for HeLa/B5 and the parental line (HeLa/0). Relative to thymidine uptake by HeLa/0 cells, that by HeLa/B5 cells was distinctly less sensitive to nitrobenzlthionosine (NBMPR), a specific inhibitor of nucleoside transport in various types of animal cells. Despite this difference in NBMPR sensitivity, both cell lines possessed the same number of high affinity NBMPR binding sites per mg cell protein. The altered kinetics of thymidine uptake and the NBMPR insensitivity of that function in HeLa/B5 cells suggest that resistance to BUdR is due to an altered thymidine transport mechanism.     

Effect of protease inhibitors on protein degradation in rat hepatoma cells I. Effect on general protein degradation

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).     

Regulatory role of endogenous regucalcin in the enhancement of nuclear deoxyribonuleic acid synthesis with proliferation of cloned rat hepatoma cells (H4-II-E)

The role of endogenous regucalcin in the regulation of deoxyribonuleic acid (DNA) synthesis in the nuclei of the cloned rat hepatoma cells (H4-II-E) with proliferative cells was investigated. Cells were cultured for 6-96 h in a alpha-minimum essential medium (alpha-MEM) containing fetal bovine serum (FBS; 1 or 10%). Cell number was significantly increased between 24 and 96 h after culture with 10% FBS; cell proliferation was markedly stimulated by culture with 10% FBS as compared with that of 1% FBS. In vitro DNA synthesis activity in the nuclei of cells was significantly elevated 6 h after culture with 10% FBS and its elevation was remarkable at 12 and 24 h after the culture. Nuclear DNA synthesis activity was significantly reduced in the presence of various protein kinase inhibitors (PD98059, staurosprine, or trifluoperazine) in the reaction mixture containing the nuclei of cells cultured for 12 and 24 h with FBS (1 and 10%). The addition of regucalcin (10(-7) and 10(-6)M) in the reaction mixture caused a significant inhibition of nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (25-100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS resulted in a significant increase in nuclear DNA synthesis activity. This increase was completely blocked by the addition of regucalcin (10(-6) M). The effect of anti-regucalcin antibody (100 ng/ml) in increasing nuclear DNA synthesis activity was significantly inhibited in the presence of various protein kinase inhibitors. DNA synthesis activity was significantly enhanced in the presence of anti-regucalcin antibody (100 ng/ml) in the reaction mixture containing the nuclei of cells cultured for 24 h with 10% FBS in the presence of Bay K 8644 (2.5 x 10(-6) M). Culture with Bay K 8644 did not cause a significant increase in DNA synthesis activity in the absence of anti-regucalcin antibody. The present study demonstrates that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis with proliferative cells.     

Reciprocal control of apoptosis and proliferation in cultured rat hepatoma arl-6 cells: Roles of nutrient supply, serum, and oxidative stress

Summary In order to understand how cancer cells accumulate, rat hepatoma ARL-6 cells were cultured for 8 d to identify factors involved in spontaneous cell proliferation and apoptosis. With increasing time in culture, the proportion of cells in the proliferative phases of the cell cycle and the rate of deoxyribonucleic acid (DNA) synthesis decreased. The waning of proliferation was associated with a gradual reduction of cell viability, and this was temporally related to the appearance of typical apoptotic morphology and DNA laddering. Medium replacement or supplementation with fetal calf serum (FCS) suppressed apoptosis, while medium change, but not fetal calf serum alone, enhanced cell proliferation. Apoptosis was also suppressed by dimethyl sulfoxide (DMSO), but supplementary glutathione was without effect. Expression of poly(adenosine diphosphate[ADP]-ribose)polymerase peaked on days 3–4 of culture, and was followed by a progressive decrease thereafter, consistent with proteolytic cleavage. This decrease was prevented to varying extents by complete medium replacement, FCS and DMSO, indicating a close temporal relationship between poly(ADP-ribose)polymerase activation and apoptosis. Expression of Fas and Bcl-2 did not change appreciably over the 8-d culture, but there was a gradual increase in Bax expression; medium change, FCS and DMSO all partly inhibited Bax expression. These data indicate that spontaneous apoptosis in cultured ARL-6 cells is inversely related to cell proliferation, and that nutrient supply, and to a lesser extent, serum-derived factors and oxidative stress modulate apoptosis in this system. Proteolytic cleavage of poly(ADP-ribose)polymerase and expression of Bax are likely to be mechanistically involved with the control of spontaneous apoptosis in ARL-6 cells, whereas changes in the levels of Fas and Bcl-2 do not play a role.     

Characterization of aldehyde dehydrogenase from HTC rat hepatoma cells

We have proposed developing rat hepatoma cell lines as an in vitro model for studying the regulation of changes in aldehyde dehydrogenase activity occurring duringhepatocarcinogenesis. Aldehyde dehydrogenase purified in a single step from HTC rat hepatoma cells is identical to the aldehyde dehydrogenase isolated from rat hepatocellular carcinomas. HTC aldehyde dehydrogenase is a 110 kDa dimer composed of 54-kDa subunits, prefers NADP⁺ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes but not phenylacetaldehyde. The substrate and coenzyme specificity, effects of disulfiram, pH profile and isoelectric point of HTC aldehyde dehydrogenase are also identical to these same properties of the tumor aldehyde dehydrogenase. In immunodiffusions, both isozymes are recognized with complete identity by anti-HTC aldehyde dehydrogenase antibodies. Having established that HTC aldehyde dehydrogenase is very similar, if not identical, to the aldehyde dehydrogenase found in hepatocellular carcinomas, simplifies the development of molecular probes for examination of the regulation of tumor aldehyde dehydrogenase activity in vivo and in vitro.     

Inhibition of thymidine kinase activity by hydroxyurea during highly and less proliferative regions of rat brain

Hydroxyurea, when injected intraperitoneally, exerted marked inhibition on the activity of thymidine kinase in 5 day old postnatal cerebellum and 15 day old embryonic cerebrum. However, it failed to show any sustained inhibition on thymidine kinase activity in 5 day old postnatal cerebrum. In this case, the marginal decrease of thymidine kinase activity noticed during early intervals reversed back to more than normal value at a later time interval. These results along with our earlier findings are taken to indicate the differential action of this drug on thymidine kinase activity in rapidly and slowly proliferating regions of rat brain     

Induction of sister chromatid exchanges by benzidine in rat and human hepatoma cell lines and inhibition by indomethacin

The genotoxic activity of benzidine was studied in two cell lines derived from rat (H4) and human (HepG2) hepatomas which have been shown to be capable of activating certain promutagens. The responses were compared to results in two lung-derived fibroblast lines (IMR-90 and V79) which appear to have little or no metabolizing capability. Benzidine was found to induce sister chromatid exchanges in the two liver-derived cell lines in a dose-dependent fashion but failed to induce sister chromatid exchanges in the fibroblast lines. Since one proposed pathway for benzidine activation involves prostaglandin-mediated metabolism, we tested the effect of pretreatment with indomethacin, an inhibitor of this metabolic pathway. Indomethacin was highly effective in inhibiting benzidine-induced sister chromatid exchanges in both H4 and HepG2 cells. These results suggest that some DNA damage may occur in the livers of fast acetylating species such as the rat without prior N-acetylation and that some amount of DNA damage may occur in the livers of slow acetylating species, even when the liver is not the target organ for carcinogenesis.Abbreviations RI replication index - SCE sister chromatid exchanges     

Activation and inactivation of thyroxine by cultured rat hepatoma cells

The metabolism of thyroxine, 3,3′,5-triiodothyronine and 3,3′,5′-triiodothyronine was investigated in rat hepatoma cell cultures (R117-21B). These iodothyronines were labeled with ¹²⁵I in the phenolic ring and the metabolites were analyzed by ion-exchange column chromatography.When thyroxine was incubated with the cells at 37°C, its glucuronide was the major product and a little increase in ¹²⁵I^? was detected. Although 3,3′,5-triiodothyronine was not observed in the incubation medium, this metabolite was clearly identified in the ethanol extract obtained from the cell homogenates after 24 h incubation.This cell line also metabolized labeled 3,3′,5-triiodothyronine added to culture medium. After 24 h incubation, 3,3′,5-triiodothyronine glucuronide was the major metabolite and iodothyronine sulfates were also formed. The sulfates contained, 3,3′,5-triiodothyronine and 3,3′-diiodothyronine sulfates and an unknown component.In the metabolism of 3,3′,5′-triiodothyronine, the cells were very active in carrying out glucuronidation and phenolic ring deiodination, and this metabolism yielded 3,3′,5′-triiodothyronine and 3,3′-diiodothyronine glucuronides. The iodide fraction contained a small amount of 3,3′-diiodothyronine sulfate.These results show that the R117-21B rat hepatoma cells metabolize the thyroid hormones and their analogs by phenolic and nonphenolic ring deiodinations, by glucuronidation and by sulfation.     

The modulation of radiation-induced cell death by genistein in K562 cells：Activation of thymidine kinase 1

Ionizing radiation is one of the most effective tools in cancer therapy. In a previous study, we reported that protein tyrosine kinase (PTK) inhibitors modulate the radiation responses in the human chronic myelogenous leukemia (CML) cell line K562. The receptor tyrosine kinase inhibitor, genistein, delayed radiation-induced cell death, while non-recepter tyrosine kinase inhibitor, herbimycin A (HMA) enhances radiation-induced apoptosis. In this study, we focused on the modulation of radiation-induced cell death by genistein and performed PCR-select suppression subtractive hybridization (SSH) to understand its molecular mechanism. We identified human thymidine kinase 1 (TK1), which is cell cycle regulatory gene and confirmed expression of TK1 mRNA by Northern blot analysis. Expression ofTK1 mRNA and TK 1 enzymatic activity were parallel in their increase and decrease. TK1 is involved in G1-S phase transition of cell cycle progression. In cell cycle analysis, we showed that radiation induced G2 arrest in K562 cells but it was not able to sustain. However, the addition of genistein to irradiated cells sustained a prolonged G2 arrest up to 120 h. In addition, the expression of cell cycle-related proteins, cyclin A and cyclin B 1, provided the evidences of G I/S progression and G2-arrest, and their relationship with TKI in cells treated with radiation and genistein. These results suggest that the activation of TK1 may be critical to modulate the radiation-induced cell death and cell cycle progression in irradiated K562 cells.     

Overexpression of regucalcin suppresses cell death and apoptosis in cloned rat hepatoma H4-II-E cells induced by lipopolysaccharide, PD 98059, dibucaine, or Bay K 8644

The effect of regucalcin, a regulatory protein in intracellular signaling pathway, on cell death was investigated by using the cloned rat hepatoma H4-II-E cells overexpressing regucalcin. The hepatoma cells (wild-type) and stable regucalcin (RC)/pCXN2 transfectants were cultured for 72 h in medium containing 10% fetal bovine serum (FBS) to obtain subconfluent monolayers. After culture for 72 h, cells were further cultured for 12-72 h in medium without FBS containing either vehicle or lipopolysaccharide (LPS; 0.1 or 1.0 microg/ml). The number of wild-type cells was significantly decreased by culture for 24 or 48 h in the presence of LPS (0.1 or 1.0 microg/ml). The effect of LPS (0.1 or 1.0 microg/ml) in decreasing the number of hepatoma cells was significantly prevented in transfectants overexpressing regucalcin. However, the culture with LPS (0.1 or 1.0 microg/ml) for 72 h caused a significant decrease in cell number of transfectants. Ca(2+)/calmodulin-dependent nitric oxide (NO) synthase activity was significantly decreased by culture with LPS (1.0 microg/ml) for 24-72 h of wild-type cells. This decrease was significantly prevented in transfectants. LPS (0.1 or 1.0 microg/ml)-induced decrease in the number of wild-type cells was significantly prevented by culture with caspase-3 inhibitor (10(-8) M). Moreover, the number of wild-type cells was significantly decreased by culture with PD 98059 (10(-6) M), dibucaine (10(-6) M), or staurosporine (10(-6) M), which is an inhibitor of various protein kinases. The effect of PD 98059 or dibucaine on the number of wild-type cells was not observed in transfectants, although the effect of staurosporine was seen in transfectants. Culture with Bay K 8644 (2.5 x 10(-6) M), an agonist of Ca(2+) entry in cells, caused a significant decrease in the number of wild-type cells. Such an effect was not seen in transfectants. The presence of LPS did not significantly decrease the number of wild-type cells in the presence of Bay K 8644. Agarose gel electrophoresis showed the presence of low-molecular-weight deoxyribonucleic acid (DNA) fragments of adherent wild-type cells cultured with Bay K 8644, and this DNA fragmentation was significantly prevented in transfectants. This study demonstrates that overexpression of regucalcin has a suppressive effect on cell death induced by LPS or various intracellular signaling-related factors.     

Characteristics and aldehyde dehydrogenase activity of four rat hepatoma cell lines produced by diethylnitrosamine-phenobarbital treatment

Summary Recent studies in our laboratory have shown that five established rat hepatoma cell lines provide a wide spectrum of tumor-associated aldehyde dehydrogenase (ALDH) activity representative of the range of activities of this enzyme seen in primary rat hepatocellular carcinomas. Four newly established rat hepatoma cell lines, RLT-2M, RLT-3C, RLT-9F, and RLT-5G, were derived from a primary hepatocellular carcinoma. The primary tumor was induced by a single injection of diethylnitrosamine (15 μM/g body weight) to a 1-d-old female S-D rat followed at weaning by chronic phenobarbital treatment. RLT-2M was established from outgrowths of minced tumor pieces. RLT-3C, RLT-9F, and RLT-5G were cloned from RLT-2M by the serial endpoint dilution. All four lines have been maintained in culture for over 100 passages. The ALDH phenotype in both the primary tumor and the four new cell lines was determined by total activity assay, gel electrophoresis, and histochemistry. By total activity assay, the primary tumor did not possess significant tumor-ALDH activity. In contrast, the four new cell lines expressed tumor-ALDH activity. However, they differed in their basal ALDH activities and in ALDH inducibility by 3-methycholanthrene, benzo(a)pyrene, and phenobarbital. Additionally, significant decreases in tumor-ALDH activity occurred when cells from each line were passaged in vivo. The four lines have been characterized by light and electron microscopic morphology, tumorigenicity, chromosome number, doubling time, and colony formation efficiency, in soft agar. This work was submitted by K.-H. L. in partial fulfillment of the requirements for the Doctor of Philosophy degree in The Graduate School of The University of Alabama. This work was supported by grant CA-21103 from the National Cancer Institute, Bethesda, MD.     

Efficient production of nuclear transferred rat embryos by modified methods of reconstruction

In this study we investigated spontaneous oocyte activation and developmental ability of rat embryos of the SD-OFA substrain. We also tried to improve the somatic cell nuclear transfer (SCNT) technique in the rat by optimizing methods for the production of reconstructed embryos. About 20% of oocytes extruded the second polar body after culture for 3 hr in vitro and 84% of oocytes were at the MII stage. MG132 blocked spontaneous activation but decreased efficiency of parthenogenetic activation. Pronuclear formation was more efficient in strontium-activated oocytes (66.1-80.9%) compared to roscovitine activation (24.1-54.5%). Survival rate after enucleation was significantly higher (89.4%) after slitting the zona pellucida and then pressing the oocyte with a holding pipette in medium without cytochalasin B (CB) compared to the conventional protocol using aspiration of the chromosomes after CB treatment (67.7%). Exposure of rat ova to UV light for 30 sec did not decrease their in vitro developmental capacity. Intracytoplasmic cumulus cell injection dramatically decreased survival rate of oocytes (42%). In contrast, 75.9% of oocytes could be successfully electrofused. Development to the 2-cell stage was reduced after SCNT (24.6% compared 94.6% in controls) and none from 244 reconstructed embryos developed in vitro beyond this stage. After overnight in vitro culture, 74.4% of the SCNT embryos survived and 56.1% formed pronuclei. The pregnancy rate of 33 recipients after the transfer of 695 of these cloned embryos was, however, very low (18.2%) and only six implantation sites could be detected (0.9%) without any live fetuses and offspring.