5-chlorodeoxycytidine sensitizes cells to x-ray and is incorporated as 5-chlorodeoxyuridine in tumor DNA

International Nuclear Information System (INIS)

Perez, L.M.; Greer, S.

1985-01-01

5-Chlorodeoxycytidine (CldC) coadministered with tetrahydrouridine (H/sub 4/U), an inhibitor of its deamination, sensitizes HEp-2 cells to X-ray and is incorporated in DNA as 5-chlorodeoxyuridine (CldU). CldC possesses a reasonable Km value (56 μM) with respect to human deoxycytidine kinase (dCK) in contrast to the low affinities of BrdC and IdC (400 and 1000μM, respectively; the Km value for dC = 2μM). Preincubation with N-phosphonacetyl-L-aspartate (PALA) and 5-fluorodeoxyuridine (FdU), inhibitors of pyrimidine biosynthesis, enhances sensitization. X-ray survival curves of HEp-2 cells treated with PALA and FdU (or FdC + H/sub 4/U) and CldC + H/sub 4/U are characterized by dose enhancement ratios of 2.5 or greater. Substantial sensitization by CldC + H/sub 4/U also occurs with Sarcoma-180 and RIF-1 cells in culture . CldC + H/sub 4/U should result in circumvention of catabolism and selective toxicity to tumors via inhibition of nucleoside reductase by CldUTP as well as selective incorporation of CldU in tumors possessing high levels of dCMP deaminase and dCK, enzymes that are markedly elevated in many human tumors. CldU, derived from CldC, is incorporated to a greater extent in the DNA of a solid tumor (S-180) than in normal tissue of the mouse. This may result in selective tumor radiosensitization

Fibre autoradiography of repair and replication in DNA from single cells: the effect of DNA synthesis inhibitors

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Ockey, C.H.

1982-04-01

DNA fibre autoradiography, after incorporation of high specific activity /sup 3/H-thymidine and /sup 3/H-deoxycytidine, has been used to investigate repair in DNA fibres from single cells following UV, or methyl-methane sulphonate (MMS) treatment. Asynchronously growing human fibroblasts, leucocytes, and HeLa cells at different phases of the cell cycle have been investigated. Isotope incorporation in repair could be differentiated from that involved in replication by the distribution and density of silver grains along the DNA fibres. Grain distribution due to repair was continuous over long stretches of the fibres and was at a low density, occasionally interspersed with short slightly denser segments. Replication labelling on the other hand, was dense and usually in short tandem segments. Repair labelling was of a similar overall density in fibres from a single cell, but differed in intensity from cell to cell. In mutagen treated Go (leucocytes) of G/sub 1/ (HeLa cells), repair labelling was not increased by the presence of the DNA inhibitors, hydroxyurea (HU) or 5-fluorodeoxyuridine (FUdR). Repair was not detectable in S cells however without the use of these inhibitors to reduce endogenous nucleoside production. FUdR enhanced the repair labelling in S cells only slightly, while HU increased it beyond that observed in UV irradiated, HU treated, G/sub 1/ cells. The intensity of repair labelling in fibres from mutagen treated S cells appears to be proportional to the degree of reduction of DNA chain elongation in replicons.

Analysis of clinical complication data for radiation hepatitis using a parallel architecture model

International Nuclear Information System (INIS)

Jackson, A.; Haken, R.K. ten; Robertson, J.M.; Kessler, M.L.; Kutcher, G.J.; Lawrence, T.S.

1995-01-01

Purpose: The detailed knowledge of dose volume distributions available from the three-dimensional (3D) conformal radiation treatment of tumors in the liver (reported elsewhere) offers new opportunities to quantify the effect of volume on the probability of producing radiation hepatitis. We aim to test a new parallel architecture model of normal tissue complication probability (NTCP) with these data. Methods and Materials: Complication data and dose volume histograms from a total of 93 patients with normal liver function, treated on a prospective protocol with 3D conformal radiation therapy and intraarterial hepatic fluorodeoxyuridine, were analyzed with a new parallel architecture model. Patient treatment fell into six categories differing in doses delivered and volumes irradiated. By modeling the radiosensitivity of liver subunits, we are able to use dose volume histograms to calculate the fraction of the liver damaged in each patient. A complication results if this fraction exceeds the patient's functional reserve. To determine the patient distribution of functional reserves and the subunit radiosensitivity, the maximum likelihood method was used to fit the observed complication data. Results: The parallel model fit the complication data well, although uncertainties on the functional reserve distribution and subunit radiosensitivy are highly correlated. Conclusion: The observed radiation hepatitis complications show a threshold effect that can be described well with a parallel architecture model. However, additional independent studies are required to better determine the parameters defining the functional reserve distribution and subunit radiosensitivity

Thymidine kinases share a conserved function for nucleotide salvage and play an essential role in Arabidopsis thaliana growth and development.

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Xu, Jing; Zhang, Lin; Yang, Dong-Lei; Li, Qun; He, Zuhua

2015-12-01

Thymidine kinases (TKs) are important components in the nucleotide salvage pathway. However, knowledge about plant TKs is quite limited. In this study, the molecular function of TKs in Arabidopsis thaliana was investigated. Two TKs were identified and named AtTK1 and AtTK2. Expression of both genes was ubiquitous, but AtTK1 was strongly expressed in high-proliferation tissues. AtTK1 was localized to the cytosol, whereas AtTK2 was localized to the mitochondria. Mutant analysis indicated that the two genes function coordinately to sustain normal plant development. Enzymatic assays showed that the two TK proteins shared similar catalytic specificity for pyrimidine nucleosides. They were able to complement an Escherichia coli strain lacking TK activity. 5'-Fluorodeoxyuridine (FdU) resistance and 5-ethynyl 2'-deoxyuridine (EdU) incorporation assays confirmed their activity in vivo. Furthermore, the tk mutant phenotype could be alleviated by nucleotide feeding, establishing that the biosynthesis of pyrimidine nucleotides was disrupted by the TK deficiency. Finally, both human and rice (Oryza sativa) TKs were able to rescue the tk mutants, demonstrating the functional conservation of TKs across organisms. Taken together, our findings clarify the specialized function of two TKs in A. thaliana and establish that the salvage pathway mediated by the kinases is essential for plant growth and development. © 2015 Institute of Plant Physiology and Ecology, SIBS, CAS New Phytologist © 2015 New Phytologist Trust.

Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase

International Nuclear Information System (INIS)

D'Anna, J.A.; Tobey, R.A.

1989-01-01

Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. There the authors report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 μg mL -1 aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression

DNA Damage during G2 Phase Does Not Affect Cell Cycle Progression of the Green Alga Scenedesmus quadricauda

Science.gov (United States)

Vítová, Milada; Bišová, Kateřina; Zachleder, Vilém

2011-01-01

DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase. PMID:21603605

DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda.

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Monika Hlavová

Full Text Available DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase.

Non-Neuronal Cells Are Required to Mediate the Effects of Neuroinflammation: Results from a Neuron-Enriched Culture System.

Science.gov (United States)

Hui, Chin Wai; Zhang, Yang; Herrup, Karl

2016-01-01

Chronic inflammation is associated with activated microglia and reactive astrocytes and plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's. Both in vivo and in vitro studies have demonstrated that inflammatory cytokine responses to immune challenges contribute to neuronal death during neurodegeneration. In order to investigate the role of glial cells in this phenomenon, we developed a modified method to remove the non-neuronal cells in primary cultures of E16.5 mouse cortex. We modified previously reported methods as we found that a brief treatment with the thymidine analog, 5-fluorodeoxyuridine (FdU), is sufficient to substantially deplete dividing non-neuronal cells in primary cultures. Cell cycle and glial markers confirm the loss of ~99% of all microglia, astrocytes and oligodendrocyte precursor cells (OPCs). More importantly, under this milder treatment, the neurons suffered neither cell loss nor any morphological defects up to 2.5 weeks later; both pre- and post-synaptic markers were retained. Further, neurons in FdU-treated cultures remained responsive to excitotoxicity induced by glutamate application. The immunobiology of the FdU culture, however, was significantly changed. Compared with mixed culture, the protein levels of NFκB p65 and the gene expression of several cytokine receptors were altered. Individual cytokines or conditioned medium from β-amyloid-stimulated THP-1 cells that were, potent neurotoxins in normal, mixed cultures, were virtually inactive in the absence of glial cells. The results highlight the importance of our glial-depleted culture system and identifies and offer unexpected insights into the complexity of -brain neuroinflammation.

Hypoxia-targeting antitumor prodrugs and photosensitizers

International Nuclear Information System (INIS)

Zhang Zhouen; Nishimoto, S.I.

2006-01-01

Tumor hypoxia has been identified as a key subject for tumor therapy, since hypoxic tumor cells show resistance to treatment of tumor tissues by radiotherapy, chemotherapy and phototherapy. For improvement of tumor radiotherapy, we have proposed a series of radiation-activated prodrugs that could selectively release antitumor agent 5-fluorouracil or 5-fluorodeoxyuridine under hypoxic conditions. Recently, we attempted to develop two families of novel hypoxia-targeting antitumor agents, considering that tumor-hypoxic environment is favorable to biological and photochemical reductions. The first family of prodrugs was derived from camptothecin as a potent topoisomerase I inhibitor and several bioreductive motifs. These prodrugs could be activated by NADPH-cytochrome P450 reductase or DT-diaphorase to release free camptothecin, and thereby showed hypoxia-selective cytotoxictiy towards tumor cells. These prodrugs were also applicable to the real-time monitoring of activation and antitumor effect by fluorometry. Furthermore, the camptothecin-bioreductive motif conjugates was confirmed to show an oxygen-independent DAN photocleaving activity, which could overcome a drawback of back electron transfer occurring in the photosensitized one-electron oxidation of DNA. Thus, these camptothecin derivatives could be useful to both chemotherapy and phototherapy for hypoxic tumor cells. The second family of prodrugs harnessed UV light for cancer therapy, incorporating the antitumor agent 5-fluorourcil and the photolabile 2-nitrobenzyl chromophores. The attachment of a tumor-homing cyclic peptide CNGRC was also employed to construct the prototype of tumor-targeting photoactiaved antitumor prodrug. These novel prodrugs released high yield of 5-fluorourcil upon UV irradiation at λ ex =365 nm, while being quite stable in the dark. The photoactivation mechanism was also clarified by means of nanosecond laser flash photolysis. (authors)

A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame.

Science.gov (United States)

Sahli, R; Andrei, G; Estrade, C; Snoeck, R; Meylan, P R

2000-04-01

Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.

Tumor and liver drug uptake following hepatic artery and portal vein infusion

International Nuclear Information System (INIS)

Sigurdson, E.R.; Ridge, J.A.; Kemeny, N.; Daly, J.M.

1987-01-01

Anatomic dye injection studies of the blood supply of colorectal hepatic metastases suggest that tumors are supplied predominantly by the hepatic artery. Using 13 N amino acids with dynamic gamma camera imaging in patients with colorectal hepatic metastases, it has been shown that hepatic artery infusion results in a significantly greater nutrient delivery to tumor compared with portal vein infusion. However, direct measurements of drug levels in tumor following hepatic artery and portal vein infusion in humans have not previously been reported. Patients with metastatic colorectal cancer confined to the liver received fluorodeoxyuridine (FUdR) through the hepatic artery or through the portal vein. All patients had previously failed systemic chemotherapy. Five patients with hepatic artery catheters were matched (by age, serum lactic dehydrogenase levels, percent hepatic replacement, and tumor size) with five patients with portal vein catheters. At operation, 3 H-FUdR (1 microCi/kg) and /sup 99m/Tc-macroaggregated albumin (MAA) (6 mCi) were injected into the hepatic artery or portal vein. Liver and tumor biopsies were obtained two and five minutes later. 3 H and /sup 99m/Tc were measured per gram tissue by scintillation and gamma counting. The mean liver levels following hepatic artery infusion (23.9 +/- 11.4 nmol/g) and portal vein infusion (18.4 +/- 14.5 nmol/g) did not differ. However, the mean tumor FUdR level following hepatic artery infusion was 12.4 +/- 12.2 nmol/g, compared with a mean tumor FUdR level following portal vein infusion of 0.8 +/- 0.7 nmol/g (P less than .01). This low level of tumor drug uptake after portal vein infusion of FUdR predicts minimal tumor response to treatment via this route. Thus, regional chemotherapy for established colorectal hepatic metastases should be administered through the hepatic artery

The treatment of colorectal liver metastases with conformal radiation therapy and regional chemotherapy

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Robertson, John M; Lawrence, Theodore S; Walker, Suzette; Kessler, Marc L; Andrews, James C; Ensminger, William D

1995-05-15

Purpose: Whole-liver radiation, with or without chemotherapy, has been of modest benefit in the treatment of unresectable hepatic metastases from colorectal cancer. A Phase I/II study combining escalating doses of conformally planned radiation therapy (RT) with intraarterial hepatic (IAH) fluorodeoxyuridine (FdUrd) was performed. Methods and Materials: Twenty-two patients with unresectable hepatic metastases from colorectal cancer, 14 of whom had progressed after previous chemotherapy (2 with prior IAH FdUrd), were treated with concurrent IAH FdUrd (0.2 mg/kg/day) and conformal hepatic radiation therapy (1.5-1.65 Gy/fraction twice a day). The total dose of radiation given to the tumor (48-72.6 Gy) depended on the fraction of normal liver excluded from the high-dose volume. All patients were assessed for response, toxicity, hepatobiliary relapse, and survival. Median potential follow-up was 42 months. Results: Eleven of 22 patients demonstrated an objective response, with the remainder showing stable disease. Actuarial freedom from hepatic progression was 25% at 1 year. The most common acute toxicity was mild to moderate nausea and transient liver function test abnormalities. There were three patients with gastrointestinal bleeding (none requiring surgical intervention) after the completion of treatment. Overall median survival was 20 months. The presence of extrahepatic disease was associated with decreased survival (p < 0.01). Conclusions: Combined conformal radiation therapy and IAH FdUrd can produce an objective response in 50% of patients with hepatic metastases from colorectal cancer. However, response was not durable, and hepatic progression was frequent. Improvements in hepatic tumor control for patients with metastatic colorectal cancer may require higher doses of conformal radiation and/or improved radiosensitization. In an effort to increase radiosensitization, we have recently initiated a clinical trial combining IAH bromodeoxyuridine, a thymidine analog

Auger electron emitter against multiple myeloma - targeted endo-radio-therapy with {sup 125}I-labeled thymidine analogue 5-iodo-4'-thio-2'-deoxyuridine

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Morgenroth, Agnieszka, E-mail: amorgenroth@ukaachen.de [Nuclear Medicine Clinic, University Ulm, Albert-Einstein-Allee 23, D-89081 Ulm (Germany); Nuclear Medicine Clinic, University Aachen, RWTH, Pauwelsstrasse 30, D-52074 Aachen (Germany); Dinger, Cornelia; Zlatopolskiy, Boris D.; Al-Momani, Ehab; Glatting, Gerhard [Nuclear Medicine Clinic, University Ulm, Albert-Einstein-Allee 23, D-89081 Ulm (Germany); Mottaghy, Felix M. [Nuclear Medicine Clinic, University Aachen, RWTH, Pauwelsstrasse 30, D-52074 Aachen (Germany); Reske, Sven N. [Nuclear Medicine Clinic, University Ulm, Albert-Einstein-Allee 23, D-89081 Ulm (Germany)

2011-10-15

Introduction: Multiple myeloma (MM) is a plasma cell malignancy characterized by accumulation of malignant, terminally differentiated B cells in the bone marrow. Despite advances in therapy, MM remains an incurable disease. Novel therapeutic approaches are, therefore, urgently needed. Auger electron-emitting radiopharmaceuticals are attractive for targeted nano-irradiation therapy, given that DNA of malignant cells is selectively addressed. Here we evaluated the antimyeloma potential of the Auger electron-emitting thymidine analogue {sup 125}I-labeled 5-iodo-4'-thio-2'-deoxyuridine ([{sup 125}I]ITdU). Methods: Cellular uptake and DNA incorporation of [{sup 125}I]ITdU were determined in fluorodeoxyuridine-pretreated KMS12BM, U266, dexamethasone-sensitive MM1.S and -resistant MM1.R cell lines. The effect of stimulation with interleukin 6 (IL6) or insulin-like growth factor 1 (IGF1) on the intracellular incorporation of [{sup 125}I]ITdU was investigated in cytokine-sensitive MM1.S and MM1.R cell lines. Apoptotic cells were identified using Annexin V. Cleavage of caspase 3 and PARP was visualized by Western blot. DNA fragmentation was investigated using laddering assay. Therapeutic efficiency of [{sup 125}I]ITdU was proven by clonogenic assay. Results: [{sup 125}I]ITdU was shown to be efficiently incorporated into DNA of malignant cells, providing a promising mechanism for delivering highly toxic Auger radiation emitters into tumor DNA. [{sup 125}I]ITdU had a potent antimyeloma effect in cell lines representing distinct disease stages and, importantly, in cell lines sensitive or resistant to the conventional therapeutic agent, but was not toxic for normal plasma and bone marrow stromal cells. Furthermore, [{sup 125}I]ITdU abrogated the protective actions of IL6 and IGF1 on MM cells. [{sup 125}I]ITdU induced massive damage in the DNA of malignant plasma cells, which resulted in efficient inhibition of clonogenic growth. Conclusion: These studies may provide a

Auger electron emitter against multiple myeloma - targeted endo-radio-therapy with 125I-labeled thymidine analogue 5-iodo-4'-thio-2'-deoxyuridine

International Nuclear Information System (INIS)

Morgenroth, Agnieszka; Dinger, Cornelia; Zlatopolskiy, Boris D.; Al-Momani, Ehab; Glatting, Gerhard; Mottaghy, Felix M.; Reske, Sven N.

2011-01-01

Introduction: Multiple myeloma (MM) is a plasma cell malignancy characterized by accumulation of malignant, terminally differentiated B cells in the bone marrow. Despite advances in therapy, MM remains an incurable disease. Novel therapeutic approaches are, therefore, urgently needed. Auger electron-emitting radiopharmaceuticals are attractive for targeted nano-irradiation therapy, given that DNA of malignant cells is selectively addressed. Here we evaluated the antimyeloma potential of the Auger electron-emitting thymidine analogue 125 I-labeled 5-iodo-4'-thio-2'-deoxyuridine ([ 125 I]ITdU). Methods: Cellular uptake and DNA incorporation of [ 125 I]ITdU were determined in fluorodeoxyuridine-pretreated KMS12BM, U266, dexamethasone-sensitive MM1.S and -resistant MM1.R cell lines. The effect of stimulation with interleukin 6 (IL6) or insulin-like growth factor 1 (IGF1) on the intracellular incorporation of [ 125 I]ITdU was investigated in cytokine-sensitive MM1.S and MM1.R cell lines. Apoptotic cells were identified using Annexin V. Cleavage of caspase 3 and PARP was visualized by Western blot. DNA fragmentation was investigated using laddering assay. Therapeutic efficiency of [ 125 I]ITdU was proven by clonogenic assay. Results: [ 125 I]ITdU was shown to be efficiently incorporated into DNA of malignant cells, providing a promising mechanism for delivering highly toxic Auger radiation emitters into tumor DNA. [ 125 I]ITdU had a potent antimyeloma effect in cell lines representing distinct disease stages and, importantly, in cell lines sensitive or resistant to the conventional therapeutic agent, but was not toxic for normal plasma and bone marrow stromal cells. Furthermore, [ 125 I]ITdU abrogated the protective actions of IL6 and IGF1 on MM cells. [ 125 I]ITdU induced massive damage in the DNA of malignant plasma cells, which resulted in efficient inhibition of clonogenic growth. Conclusion: These studies may provide a novel treatment strategy for overcoming

Dependence of the rate of DNA synthesis in x-irradiated HeLa S3 cells on dose and time after exposure

International Nuclear Information System (INIS)

Tolmach, L.J.; Jones, R.W.

1977-01-01

After irradiation of randomly dividing cultures of HeLa S3 cells with 220-kV x rays, the rate of DNA synthesis, measured by pulsed incorporation of labeled thymidine, falls nearly exponentially with time (t/sub 1/2/ approximately 1.3 hr), in a dose-independent fashion. The fall is less rapid than that observed after addition of inhibitors of protein synthesis. With doses up to 8 krad, the rate reaches a minimum and begins to increase after 1-3 hr, the minima occurring at lower values and at slightly later times with increasing dose. The increase appears to be roughly linear for about 6 hr, with the slope an inverse function of dose in the range 1-8 krad. About 7-9 hr after the completion of irradiation, the rate again falls, although no more than 10 percent of the cells die sooner than 14 hr after irradiation with 8 krad (and later with smaller doses). Fluorodeoxyuridine-mediated delay in expression of the depression, described previously for doses up to 1 krad, occurs also at higher doses. During the period when the rate per culture rises, the rate in the individual cells, measured autoradiographically, appears to increase also, i.e., the rise presumably does not merely reflect populational shifts. The initial descending portion of the rate curve can be at least partially separated from the ascending portion by administering the total dose in suitably spaced fractions. If interpreted in terms of the model that attributes the initial depression in rate of synthesis to a temporary absence of replicon initiation, the results indicate that initiation is halted by an x-ray dose smaller than 1 krad; that it begins again after a dose-dependent delay amounting to about 0.7 hr after 1 krad and 1.5 hr after 7 krad; and that once begun, the rate of synthesis increases in a dose-dependent fashion. The second depression might derive from synchronization and/or from the imminence of cell death

Kinetic and photochemical studies and alteration of ultraviolet sensitivity of Escherichia coli thymidine kinase of halogenated allosteric regulators and substrate analogues

International Nuclear Information System (INIS)

Chen, M.S.; Prusoff, W.H.

1977-01-01

The effect of various halogenated and nonhalogenated allosteric effectors on the sensitivity of Escherichia coli thymidine kinase to ultraviolet radiations (uv,253.7 nm) was investigated. All naturally occurring dNTPs convert the monomeric form of the enzyme into the dimeric form which is less sensitive to uv inactivation. Whereas 5-iodo-2'-deoxycytidine triphosphate (IdCTP) and 5-iodo-2' deoxyuridine triphosphate (IdUTP) enhance the uv inactivation of the enzyme, 5-bromo-2'-deoxyuridine triphosphate and 5-bromo-2'-deoxycytidine triphosphate exert a protective effect similar to that produced by the corresponding naturally occurring effectors, dTTP and dCTP. The enhanced uv inactivation by IdUTP is prevented totally by dTTP, but only partially by dCTP or dThd, whereas the enhanced sensitization by IdCTP is prevented almost totally by dCTP, partially by dTTP, and not at all by dThd. The uv sensitization of thymidine kinase by IdCTP appears to be at the regulatory site since a maximum saturation effect is observed, and the concentration required to exert a 50% maximal uv sensitization is similar to its K/sub m/ for enhancement of catalytic activity. When the enzyme was irradiated in the presence of either [2- 14 C]IdUTP or [2- 14 C]IdUrd, zone sedimentation analysis in sucrose density gradients showed the sedimentation coefficient of the radioactive labeled proteins to be the same, 3.8 S. Hence uv irradiation of the effector-induced dimer resulted in not only dissociation to the monomer, but also complete loss of catalytic activity. The substitution of an azido group for the 5'-OH group of 5-iodo-, 5-bromo-, 5-chloro-, or 5-fluorodeoxyuridine greatly decreased their affinity for thymidine kinase, and in addition the kinetics of inhibition changed from a competitive to a noncompetitive pattern. The presence of the azido moiety in the 5' position of the halogenated nucleosides did not enhance the rate of uv inactivation of the enzyme