Birgitte Munch-Petersen

The deoxyribonucleoside 5′-triphosphate (dATP and dTTP) pool in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Abstract The pool size of dATP and dTTP in human lymphocytes was studied in untreated and PHA-treated cells. Different methods of extracting the cellular content of dATP and dTTP have been investigated and extraction with 60% methanol was preferred. The pool size of dATP and dTTP in non-stimulated lymphocytes was about 0.2 and 0.05 pmoles/106 cells, respectively. After treatment with PHA for about 50 h the dATP and dTTP pools reached peak values representing increases in the pools of 20 and 170 fold, respectively. The variation in the pool sizes during transformation was paralleled by the variation of the rate of incorporation of labeled deoxy-thymidine into cellular DNA.

Differences in the kinetic properties of thymidine kinase isoenzymes in unstimulated and phytohemagglutinin-stimulated human lymphocytes

SummaryThe two thymidine kinases, TK 1 and TK 2, found in phytohemagglutinin-stimulated human lymphocytes and the thymidine kinase, TK 2N, found in unstimulated human lymphocytes were purified and characterized. All three kinases had molecular weights between 70000 and 75000 which increased to 170000–200000 in the presence of 2 mM ATP.Studies on the kinetic properties of the enzymes with thymidine and ATP as the substrates and dTTP as the inhibitor showed clear differences between TK 1 and TK 2, but a close similarity between TK 2 and TK 2N. With thymidine as the variable substrate, TK 1 showed Michaelis-Menten kinetics, whereas TK 2 and TK 2N showed characteristic biphasic kinetics. With ATP as the variable substrate, all three enzymes showed positive cooperative kinetics, but TK 2 and TK 2N lost the cooperativity in the presence of dTTP. The results from inhibition studies showed, that dTTP was a cooperative inhibitor of TK 1 but a non-cooperative inhibitor of TK 2 and TK 2N.

The nevoid basal cell carcinoma syndrome: Sensitivity to ultraviolet and x-ray irradiation

Demographic studies in patients with skin cancer have demonstrated the importance of exposure to ultraviolet and x-ray irradiation. This paper describes in vitro studies in peripheral lymphocytes from three patients with the nevoid basal cell carcinoma syndrome. Particular stress was placed on the following factors: (1) the distribution of the lymphocyte subsets, (2) the frequency of spontaneous sister chromatid exchange, (3) the effect of ultraviolet C (UVC) (254 nm) on deoxyribonucleic acid (DNA) synthesis, (4) the effect of UVC on the phytohemagglutinin-stimulated lymphocyte proliferation, and (5) the capacity to repair x-ray-induced DNA damage. Our data indicate that the distribution of the peripheral lymphocytes was normal, while the frequency of spontaneous sister chromatid exchange was high. The capacity of the lymphocytes to repair x-ray-induced DNA damage was low in all three patients. In two patients the UVC-induced DNA synthesis was reduced, while an increased UVC-induced inhibition of lymphocyte proliferation was observed. These cellular responses in vitro to ultraviolet and x-ray irradiation correspond to the clinical features of the nevoid basal cell carcinoma syndrome. A clearly defective in vitro cellular response to x-ray irradiation, reflecting the clinically evident x-ray sensitivity in the nevoid basal cell carcinoma syndrome, has not been reported previously.

Increased number of circulating suppressor T-Lymphocytes in Sun-induced multiple skin cancers

The distribution of peripheral lymphocyte subsets was studied in fifteen patients with multiple nonmel‐anoma skin cancers, selected according to history of ultraviolet (UV) or X‐ray exposure. The skin cancer was associated with previous heavy exposure to UV light in seven patients, and past exposure to x‐rays in eight patients. In the UV group, the helper T‐lymphocytes/suppressor T‐lymphocytes (Th/Ts) ratio was abnormally low (P < 0.01) compared with the ratios of the x‐ray and control groups. The low Th/Ts ratio was associated with an absolute increase in the number of Ts. This suggests that heavy sun exposure may cause a permanent increase in the number of Ts in certain persons. These extra T‐lymphocytes may in turn prevent immune rejection of transformed keratinocytes.

DNA polymerase activity in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Abstract Requirements and optimal conditions have been studied for the activity of DNA polymerase from phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Differences were found in thermal stability and inhibitory effect of KC1 and p -chloromercuribenzoate. The relationship was determined between DNA polymerase activity, cellular pools of dATP, dTTP and incorporation of deoxythymidine into DNA during transformation. The increase in polymerase activity was paralleled by a similar increase in the pools of dATP and dTTP. The enzyme activity and the pool sizes of both nucleotides reached a maximum simultaneously with the peak of deoxythymidine incorporation into DNA. Studies in which protein synthesis was limited by cycloheximide showed that both the DNA polymerase activity and the rise in the pool sizes of both nucleotides were abolished. This implies that the de novo synthesis is required for the enzymes involved.

X-ray and UV-radiation sensitivity of circulating lymphocytes in multiple epidermal cancer in relation to previous radiation exposure

The cellular sensitivity to X rays (200 kV, 16 mA) and UV radiation (254 nm) was examined in lymphocytes from three groups of patients with multiple epidermal malignant tumors, selected by their clinical history of carcinogenesis. Eight patients previously exposed to low energy ionizing radiation (less than or equal to 12 kV) had an increased cellular sensitivity to UV radiation as well as X rays compared with 24 age and sex matched controls. This indicates the existence of a cellular cross-sensitivity to UV radiation and ionizing radiation not previously established for human cells. In contrast six patients previously exposed to high energy ionizing radiation (between 25 and 170 kV) had normal cellular response to both UV radiation and X rays, indicating a different biologic effect of low and high energy ionizing radiation. In the third group of patients, previously exposed to therapeutic UV radiation/excess sunlight, the lymphocytes had a normal response to X rays, but an increased sensitivity to UV radiation. The possibility of evaluating the individual risk at radiation exposure is suggested.

Thymidine kinase in human leukemia expression of the lymphoblastic isoenzyme in three patients with acute myelocytic leukemia

The dominating thymidine kinase activity in mononuclear white blood cells from three patients with untreated acute myelocytic leukemia (AML) was compared with TK 1 from phytohemagglutinin-stimulated and TK 2 from unstimulated, normal lymphocytes. The enzyme activity in the AML cells and the stimulated lymphocytes was found to be in the same range. Regarding the combined thymidine and dTTP kinetics, the enzymes from the three AML patients resembled TK 1, but the ATP kinetics were different and the molecular weights were lower, as previously found for thymidine kinases from other leukemic cells. Therefore, the designation TK-1-onc is suggested for the thymidine kinases from the AML cells.

Thymidine kinase isoenzymes in human acute and chronic lymphatic leukemia

The dominating thymidine kinase isoenzyme was examined in mononuclear leucocytes from two patients, one with acute and one with chronic lymphatic leukemia. The two isoenzymes exhibited Michaelis-Menten substrate kinetics with ATP and cooperative inhibition kinetics with dTTP. The substrate kinetics with thymidine were different. According to the enzymatic properties the isoenzymes from the acute and chronic lymphatic cells were designated TK 3 and TK 4, respectively. Comparison with the isoenzymes in normal lymphocytes (TK 1, TK 2) and in acute monocytic leukemic cells (TK 3, TK 4) indicated the existence of three thymidine kinase isoenzymes in human leukemic cells which differed from the two isoenzymes in normal human lymphocytes.

Thymidine kinase isoenzymes in human acute monocytic leukemia.

SummaryTwo thymidine kinase isoenzymes, TK 3 and TK 4, from mononuclear leucocytes from a patient with acute monocytic leukemia, were purified and characterized in regard to the molecular weights and kinetic properties.The molecular weights of TK 3 and TK 4 were 60 000 and 45 000, respectively. In the presence of 2 mM ATP, the molecular weight of TK 3 increased to 200 000, whereas the molecular weight of TK 4 was unchanged.Studies of the kinetic properties showed clear differences between TK 3 and TK 4. With thymidine as substrate, TK 3 showed biphasic kinetics with a Km of 22 µM, and TK 4 showed Michaelis-Menten kinetics with a Km of 0.33 µM With ATP as substrate, TK 3 showed Michaelis-Menten kinetics with a Km of 100 µM, and TK 4 showed biphasic kinetics with a Km of 3.5 µM. With dTTP as inhibitor, TK 3 showed cooperative inhibition kinetics, and TK 4 showed non-cooperative competitive inhibition kinetics. The dTTP concentration at 50% inhibition was 75 µM for TK 3 but 380 µM for TK 4.Comparison of the molecular weights and the kinetic properties of TK 3 and TK 4 with the corresponding data previously obtained for TK 1 and TK 2 from normal human lymphocytes indicate the existence of four thymidine kinase isoenzymes in human leucocytes.

Deoxycytidylate deaminase activity in non-stimulated and phytohemagglutinin-stimulated human lymphocytes, and in leukemic cells

Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained.At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP.At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.