Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

E2F mediates induction of the Sp1-controlled promoter of the human DNA polymerase epsilon B-subunit gene POLE2.

The B-subunits of replicative DNA polymerases from Archaea to humans belong to the same protein family, suggesting that they share a common fundamental function. We report here the gene structure for the B-subunit of human DNA polymerase epsilon (POLE2), whose expression and transcriptional regulation is typical for replication proteins with some unique features. The 75 bp core promoter region, located within exon 1, contains an Sp1 element that is a critical determinant of promoter activity as shown by the luciferase reporter, electrophoretic mobility shift and DNase I footprinting assays. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Binding sites for E2F1 and NF-1 reside immediately downstream from the core promoter region. Our results suggest that human POLE2 is regulated by two E2F-pocket protein complexes, one associated with Sp1 and the other with NF-1. So far, only one replicative DNA polymerase B-subunit gene promoter, POLA2 encoding the B-subunit of DNA polymerase alpha, has been characterized. Mitogenic activation of the POLE2 promoter by an E2F-mediated mechanism resembles that of POLA2, but the regulation of basal promoter activity is different between these two genes.

Hydroxyurea-induced cell death as related to cell cycle in mouse and human T-lymphoma cells.

The association between DNA precursor synthesis, cell cycle perturbations, and cell death caused by the anticancer drug hydroxyurea was investigated in mouse and human T-lymphoma cells. Hydroxyurea inhibits the enzyme ribonucleotide reductase, leading to decreased deoxyribo nucleoside triphosphate pools and an accumulation of cells in early S-phase of the cell cycle. We wished to clarify the mechanism of cell death caused by hydroxyurea in concentrations that can be obtained therapeutically. At a 60-microM concentration of the drug, giving 25% growth inhibition during 24 h, no increase in the number of dead cells was observed as determined by cell flow calculations and density gradient centrifugation. However, the removal of hydroxyurea led to 10-30% cell loss during the following 12-h period. In parallel, there was an increase in DNA precursor levels and a rapid progression of cells through S- and G2 phases of the cell cycle. The isolated dead cells showed no overrepresentation of any cell cycle phase. The results demonstrate that, although the toxic effects of low concentrations of hydroxyurea are minimal, the drug-induced unbalanced growth state can cause substantial cell death during a posttreatment period.

Selenite and selenate inhibit human lymphocyte growth via different mechanisms.

Selenium compounds like selenite and selenate have strong inhibitory effects, particularly on mammalian tumor cell growth by unknown mechanisms. We found that the addition of sodium selenite and sodium selenate inhibited the growth of human 3B6 and BL41 lymphocytes. Selenite was more potent because 10 microM selenite produced a growth inhibitory effect similar to that of 250 microM selenate. The mechanism of action of selenite and selenate appears to be different. 3B6 and BL41 cells treated with selenite accumulated in the S-phase; however, selenate caused an accumulation of cells in G2. Selenite-mediated growth inhibition was irreversible, although the effects of selenate could be reversed. Selenite, in contrast to selenate, is efficiently reduced by the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). At concentrations required to observe a similar effect on cell growth, the activity of thioredoxin reductase, recently shown to be a selenoprotein, increased in selenite-treated cells and decreased in selenate-treated cells. Ribonucleotide reductase activity was inhibited in an in vitro assay by selenite and selenodiglutathione but not by selenate. These results show that selenite and selenate use different mechanisms to inhibit cell growth.

Effects of foscarnet on the cell kinetics of Madin-Darby canine kidney cells.

The antiherpes compound, foscarnet (trisodium phosphonoformate), showed concentration-dependent effects on the cell kinetics of Madin-Darby canine kidney cells. At 1 mM, only minor effects could be seen on cell proliferation and cell cycle distribution, as measured by flow cytometry DNA analysis. Treatment with 5 mM foscarnet resulted in an accumulation of cells in the S-phase although no complete cell cycle block was evident. At 10 mM foscarnet, cells accumulated earlier in the S phase, probably at the G1/S border. However, at both 5 and 10 mM foscarnet the block was not established until after 15 h incubation. Upon removing 10 mM foscarnet after 24 h incubation, G1 cells rapidly entered the S phase, whereas the progression through S and G2 + M was delayed considerably. The DNA synthesizing S phase seems, therefore, to be the main cell cycle phase affected by foscarnet.

Arginase, an S-phase enzyme in a human cell line.

Suspension cultures of 'Chang liver' cells were synchronized by preincubation in a glutamine-deficient medium or by thymidine blockade. Specific arginase activity varied in the synchronized cultures, being high when the number of S-phase cells was maximal. A relationship between high arginase activity and a high percentage of (S + G2) cells was also found when unsynchronized cells were separated by velocity sedimentation. The increase in arginase activity near the G2/S border was totally inhibited in the presence of cycloheximide. The rate of decrease in activity after addition of the drug indicated that the variations in arginase activity during the mitotic cycle were the result of variations in the rate of synthesis of the enzyme, while the rate of degradation was more or less constant, corresponding to 4--6% per h. The role of arginase in cells lacking a urea cycle and the regulation of arginase activity in 'Chang liver' cells is discussed.

Existence of phosphorylated and dephosphorylated forms of cytosolic thymidine kinase (TK1).

In this study we examine whether different TK1 variants of pI 6.9 and 8.3 found by isoelectric focusing gel electrophoresis (IFE) reflect just a phenotype difference due to phosphorylation modifications or have a real phenotypic background. The phosphorylation degree of purified TK1 variants was analyzed by determining the changes in the pI values after treatment with alkaline phosphatase, using IFE. The genetic origin of the two TK1 variants was studied by determining their mol wt. by means of SDS-gelelectrophoresis. Furthermore, the subcellular distribution of the two TK1 variants was also studied. Alkaline phosphatase treatment changed the pI value of purified TK1 from 6.9 to 8.3. No change in the pI value was found when purified TK1 corresponding to pI 8.3 was treated in the same way. Similar results were obtained when treated a cytosolic fraction with alkaline phosphatase. Antibody raised against the C-terminal part of human TK1 only recognized the dephosphorylated TK1 variant corresponding to pI 8.3. There was no difference in the molecular weight between the two TK1 variants. Thus, we concluded that the TK1 variants corresponding to pI 6.9 and 8.3 are of the same genetic origin, but consist of phosphorylated and dephosphorylated forms.

Serum thymidine kinase 1 is a prognostic and monitoring factor in patients with non-small cell lung cancer.

Thymidine kinase 1 (TK1) is a pyrimidine metabolic pathway enzyme involved in salvage DNA synthesis and thus, a cell cycle-dependent marker. We have developed anti-TK1 monoclonal/polyclonal Abs raised against a 15-amino acid synthetic peptide (KPGEAVAARKLFAPQ) corresponding to part of the C-terminus of human TK1. These Abs are useful for both serological and immunohistochemical detection of TK1 in a number of cancer diseases. In this study we examined TK1 concentration in serum (STK1) in 250 preoperative non-small cell lung cancer patients (NSCLC), including 188 non-metastatic (group M0) and 62 metastatic patients (group M1). Serum from 16 healthy individuals was used as controls. The concentration of STK1 of preoperative NSCLC patients was significantly higher than STK1 of healthy individuals (p<0.0001). In group M0, preoperative STK1 concentration was significantly higher in patients of tumour size T2, as compared to tumour size T1 (p=0.042), and in T3-T4, as compared to T1-T2 (p=0.01). No significant difference in STK1 concentration between patients of tumour stage I and stage II (p=0.057) were found, but significantly higher STK1 concentration in patients of stage III, as compared stage I-II (p=0.025). No significant difference of STK1 concentration were found in patients of group M1 concerning tumour size or tumour stage, or between patients with adenocarcinomas (AC) and squamous cell carcinomas (SCC). We studied the changes of STK1 concentration individually one month after operation in metastatic subjects (group M1, n=19) and in tumour-free subjects (group M0, n=38). In the M0 group, the concentration of STK1 one month after operation was significantly decline by 45%, when comparing to concentrations of STK1 preoperative (p<0.001). In the group M1, however, no significant decrease in STK1 concentrations were found one month after operation. We conclude that STK1 has prognostic value and is a reliable marker for monitoring the response to surgery of NSCLC patients.

Characterization of a peptide antibody against a C-terminal part of human and mouse cytosolic thymidine kinase, which is a marker for cell proliferation.

An affinity-purified anti-TK1 antibody (pAb1) raised against a synthetic peptide (amino acids K211PGEAVAARKLFAPQ225) corresponding to part of the C-terminus of human cytosolic thymidine kinase (TK1) was produced and characterized by enzyme-linked immunosorbent assay, Western immunoblotting and immunoprecipitation as well as by immunostaining of intact cells. pAb1 recognized a single 25 kDa TK1 polypeptide in extracts of human and rodent cells. The protein was localized to the cytoplasm, as studied by immunohistochemistry and there was no staining in G1/G0 cells or mutant cells lacking TK1 activity, while it was high in S-phase and G2 cells. When series of peptides were tested for antibody binding in which alanine was replacing each of the other amino acids one by one, lysines 211 and 220, proline 212 and glutamic acid 214 were found to be important for antibody reactivity. These results indicate that amino acids 211-214, which may form a turn region, constitute a major recognition site for pAb1, and this structure may also be involved in the cell cycle-dependent modification of TK1, pAb1 is a very useful tool for studies of the cell cycle regulation of TK1, and it may be used to identify and quantify rapidly proliferating cells such as tumor cells.

Microfilament-disrupting Clostridium difficile toxin B causes multinucleation of transformed cells but does not block capping of membrane Ig.

The effects of Clostridium difficile toxin B on some actin-dependent cellular functions were studied. A three-day incubation of intoxicated B-lymphocytes and transformed 3T3 fibroblasts resulted in dose-dependent multinucleation. Using vimentin-negative Daudi cells we showed that this effect of toxin B does not involve vimentin. As DNA and protein syntheses are not impaired in the cells used, the results suggest that toxin B has an effect on the actin-containing contractile ring during mitosis, in a manner similar to that of the microfilament-disrupting agent cytochalasin B. Toxin B is the first bacterial toxin shown to have this effect. It was also found that the capping of surface IgM on B-lymphocytes was not inhibited by toxin B, whereas cytochalasin B did inhibit capping. These results suggest that capping is dependent on a specific membrane-associated actin structure, which is not affected by toxin B.

Cell cycle related studies on thymidine kinase and its isoenzymes in Ehrlich ascites tumours.

Thymidine kinase (TK) activity was measured in relation to the cell cycle of in vivo growing ascites tumour cells. The cells were synchronized by means of centrifugal elutriation and the cell cycle composition of the cell fractions was determined by flow cytometry. TK activity was low in G1, increased during S phase and declined in G2. A half-life of TK activity of about 45 min was found throughout the cell cycle. Four isoenzymes at pI values of 4.1, 5.3, 6.9 and 8.3, denoted as isoenzymes 1-4, were identified using isoelectric focusing. Isoenzymes 3 and 4 were responsible for the profound cell cycle related changes in the TK activity. Corresponding isoenzymes were also found in the fetal mouse liver. In the adult mouse liver isoenzyme 2 was the dominating isoenzyme. The half-life of the isoenzymes was in the same range as for the total TK activity. We conclude that the low TK activity in G1 is due to degradation of the enzyme in G2 at a normal rate combined with an arrest in the synthesis of TK. We also conclude that isoenzyme 4 and the intermediate isoenzyme 3, which had earlier been suggested to be a mitochondrial form of TK, in fact represent cytoplasmatic forms of TK. According to cell cycle and pI studies, isoenzyme 2 belongs to the mitochondrial form. Studies with various phosphor donors and specific substrates, however, indicate that it also contains a cytoplasmic component.

X-irradiation effects on thymidine kinase (TK): I. TK1 and 2 in normal and malignant cells.

The effect of radiation on TK is more complicated than would be expected from earlier results on bone marrow cells (Feinendegen et al. 1984, Int. J. Radiat. Biol. 45, 205). TK activity increased at 0.01 Gy and then decreased up to 1 Gy in mouse spleen. In contrast to the results for the spleen, an increase in activity at 0.1 Gy was seen in mouse thymus. The activity of dephosphorylated TK1 (TK1a) in both spleen and thymus was reduced to 50% after irradiation at 0.5-1 Gy. The degree of phosphorylation (TK1b/TK1a ratio) changed in spleen, but not in thymus. The activity of TK2 in mouse liver increased at 3 h after 5 Gy by about 60%. In mouse ascites tumour, a dose-independent (1-5 Gy) oscillating TK1 activity was found up to 24 h, especially for TK1a and TK1b. The amount of TK1 was unchanged up to 12 h, but decreased at 24 h. This suggests that the differences in the changes in the degree of phosphorylation of TK1 after irradiation among spleen, thymus and ascites tumour further underline the complexity of the response of TK1 activity to irradiation. The dramatic change in the activities of TK1a and TK1b may illustrate that both of them are more radiosensitive than TK-h, a variant with mixed TK1 and TK2 properties.

X-irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity.

The purpose of this study was to investigate the mechanism behind the high sensitivity of thymidine kinase 1 (TK1) to X-irradiation. The deoxythymidine triphosphate (dTTP) pool was studied in mouse ascites tumour cells 1-24 h after X-irradiation with 5 Gy. Irradiation changed the Michaelis-Menten kinetics of TK1 from linear to biphasic, showing a negative co-operativity. These changes were closely related to changes in the dTTP pool. Addition of dTTP to the cell extract of non-irradiated cells, or thymidine (dTdR) to the culture medium, resulted in changes very similar to the kinetics found in the irradiated cells. Addition of 5 cent-amino-5 cent-deoxythymidine (5 cent-AdTdR), a thymidine analogue that eliminated the inhibitory effect of dTTP on TK1 activity, completely abolished the irradiation-induced inhibition of TK1 activity. We suggest that the reduced TK1 activity is mainly due to an elevated intracellular concentration of dTTP.

Cell cycle-dependent regulation of the DNA-dependent protein kinase.

Human DNA-dependent protein kinase (DNA-PK) is a nuclear-localized serine/threonine protein kinase. The holoenzyme consists of a catalytic subunit with a molecular mass of 465 kDa and a DNA-binding heterodimer Ku86/70. The kinase has been implicated in a variety of nuclear processes including V(D)J recombination, double-strand break repair, and transcription. Cells with defective DNA-PK activity show increased radiosensitivity and lack of V(D)J recombination. To study DNA-PK activity during the cell cycle, HeLa cells were separated by elutriation centrifugation into different cell cycle compartments based on cellular size. DNA-PK activity was found to vary during the cell cycle. The kinase activity was lowest during G1 phase and increased dramatically as the cells entered S phase and remained high during the G2-phase. The subcellular distribution of DNA-PKcs is relocalized from the cytoplasm during M and G1 phases to the nucleus during G1-S phase transition and S phase. Expression of both the catalytic subunit and the Ku86/70 heterodimer was found to be constant throughout the cell cycle. This study demonstrates that DNA-PK activity as well as its subcellular localization fluctuates during the cell cycle. In addition, the distribution of DNA-PK during M phase corresponds with low DNA-PK activity.

Effects of 5-fluorouracil on cell cycle arrest and toxicity induced by X-irradiation in normal mammalian cells.

UNLABELLED: From clinical studies in cancer patients and experimental in vitro studies, there is evidence of an increased cytotoxic effect, and even synergy, when irradiation is combined with 5-fluorouracil (5-FU). The mechanism for this is unclear. MATERIALS AND METHODS: Mouse fetuses (C3H) have been exposed in vivo to X-irradiation and 5-fluorouracil (5-FU) as single agents or in combination. Cell proliferation, cell cycle progression, fetal survival and incidence of fetal malformations have been studied. PURPOSE: The aim of this study was to determine possible synergistic cytotoxic effects when 5-FU and ionizing radiation were combined, particularly concerning the regulation of cell cycle progression in proliferating, non malignant mammalian cells in vivo. RESULTS: The combination of low-toxic doses of X-irradiation and 5-FU had a synergistic toxic effect in nonmalignant mouse fetuses in vivo. The cell cycle regulation was perturbed and the radiation-induced G2-arrest was eradicated by 5-FU during the initial hours. CONCLUSIONS: The time for repair of radiation induced DNA-damage is probably reduced, which may explain the increased toxicity of this combination.

Can 8-oxo-dG be used as a predictor for individual radiosensitivity?

PURPOSE: To develop predictive tests for individual radiosensitivity of tumor patients. METHODS AND MATERIALS: Acute skin reactions were clinically scored among 40 women after 46 Gy, given with 2 Gy fractions to breast and regional lymph nodes, adjuvant after surgery. The acute skin reactions were compared to the excretion of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in urine, determined by high-performance liquid chromatography (HPLC) with electrochemical detector. Specimens of urine were collected before and during postoperative radiation treatment at given intervals. We compared a group of 9 patients with the most pronounced skin reactions with another group of 8 patients with almost no skin reactions at 46 Gy. RESULTS: The level of 8-oxo-dG excreted in urine during 8 h was measured. After normalizing the excretion to irradiated volumes, dose per volume and excretion before irradiation, the 8-oxo-dG level in urine was significantly (p < 0.001) lower for the patients with pronounced skin reactions as compared to patients with minor skin reactions, at an accumulated dose of 12 Gy. In addition, the background level of 8-oxo-dG excreted before treatment started, was significantly (p = 0.043) lower for patients with minor skin reactions as compared to patients with pronounced skin reactions. The background level of 8-oxo-dG was corrected for body weight and normalized to BMI. CONCLUSION: We suggest that the excretion of 8-oxo-dG into urine of breast cancer patients is a possible marker for acute radiosensitivity.

Improved measurement of tyrosine-phosphorylated DNA-PKcs by isoelectric focusing as an indirect determination of DNA-PK activity in vivo.

UNLABELLED: Isolation of various phosphorylated forms of DNA-PKcs by SDS gel electrophoresis is difficult because of the same molecular weight. By isoelectric focusing (IEF), we confirmed a de-phosphorylated (pI 9.0) and a phosphorylated (pI 6.9) form of DNA-PKcs. When cells were damaged by X-irradiation (5 Gy), an additional tyrosine-phosphorylated (p-Tyr) DNA-PKcs form (pI 6.3) appeared in both nuclear and cytoplasmic fractions, after 30 minsutes. CONCLUSION: IEF in combination with Western blotting of p-Tyr-DNA-PKcs results in an appropriate separation of the p-Tyr form from other phosphorylated forms of DNA-PKcs, which may permit an indirect measurement of changes in DNA-PK activity in irradiated cells.

Enzyme kinetics of thymidine kinase isoenzymes of Ehrlich ascites tumour.

Three isoenzymes of thymidine kinase (TK) identified on Ehrlich ascites tumour corresponding to pl values of 5.3, and 6.9 and 8.3 were studied with respect to their kinetic properties. The isoenzymes were separated by thin-layer isoelectric focusing and measured in the presence of thymidine (TdR) as substrate, adenosinetriphosphate (ATP) as phosphor donor and deoxythymidinetriphosphate (dTTP) as inhibitor. Exponentially growing cells and growth inhibited cells were used with high proportions of the isoenzymes at pl values of 6.9 and 8.3, and 5.3 respectively. The concentrations of the former were further enhanced by the use of S-phase cells isolated by elutriator centrifugation. The isoenzymes were identified as TK1-onc, earlier found in human leukemia cells. In order to confirm the existence of TK1, particular at pl value 5.3, the ability of the isoenzymes to use different phosphor donors (ATP and uridinetriphosphate (UTP)) and substrates (TdR and deoxycytidine (dCdR) was also studied. These results showed that the isoenzymes at pl 6.9 and 8.3 correspond to TK1, while the isoenzyme at pl 5.3 is heterogeneous. Further, high resolution isoelectric focusing confirmed the existence of two isoenzymes at pl values of 5.1 and 5.3, representing mitochondrial (TK2) and cytoplasmic (TK1) isoenzymes.

Melphalan-induced DNA cross-linking and inhibition of DNA and RNA synthesis in human melanoma and lymphoblast cells.

Melphalan inhibits the incorporation of 3H-thymidine and 3H-uridine significantly more in phytohaemagglutinin (PHA)-stimulated human lymphocytes than in a human melanoma cell line (RPMI 8322). Melphalan - induced total DNA cross-linking was 1.7 times higher and DNA interstrand cross-linking was 1.8 times higher in the PHA-stimulated lymphocytes than in the melanoma cells. A higher level of DNA cross-linking was required in melanoma cells than in PHA-stimulated lymphocytes to obtain similar levels of inhibition of incorporation of 3H-thymidine and 3H-uridine. The outflow of cells from G1 to S phase was significantly more inhibited by melphalan in the lymphocytes than in the melanoma cells. Thus the melanoma cells can replicate and transcribe DNA in the presence of levels of DNA damage, which in PHA-stimulated lymphocytes strongly inhibit DNA and RNA synthesis.

A new cell proliferating marker: cytosolic thymidine kinase as compared to proliferating cell nuclear antigen in patients with colorectal carcinoma.

BACKGROUND: Proliferation markers are necessary for reliable diagnosis. Here we have presented for the first time thymidine kinase 1 (TK1) as a proliferative tumor marker for colorectal carcinoma. PATIENTS AND METHODS: Expression of TK1 in 54 colorectal lesions and 20 colorectal adenoma lesions was detected by immunohistochemistry technique (ABC). Proliferating Cell Nuclear Antigen (PCNA) was run in parallel. RESULTS: TK1-Labelling Index (LI) (65%) was higher than PCNA-LI (52%) in the malignant lesions, although not significantly different (p = 0.1717) between them. TK1-LI as well as PCNA-LI showed significant differences between colorectal carcinoma and colorectal adenoma (TK1 p = 0.0005, PCNA p = 0.0005). Both TK1-LI and PCNA were significantly different in respect to tumor stages (TK1 p = 0.0002, PCNA p = 0.0284). However, only TK1-LI showed significant difference in respect to tumor grades (p = 0.014), but not PCNA-LI (p = 0.132). CONCLUSION: TK1-LI showed more potential as a proliferating marker in colorectal carcinoma than PCNA-LI, especially for evaluating high-risk tumor grade and advanced stage in colorectal carcinoma.