David Grdina - Academia.edu (original) (raw)
Papers by David Grdina
British Journal of Cancer, Aug 1, 1983
The cytotoxic and stathomokinetic effects of vincristine (VCR) on murine fibrosarcoma (FSa) cells... more The cytotoxic and stathomokinetic effects of vincristine (VCR) on murine fibrosarcoma (FSa) cells, grown either in vitro as primary cultures or in vivo as microor macroscopic pulmonary nodules, were determined and compared. FSa cells were separated and synchronized on the basis of size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations, thus allowing determination of age-dependent cytotoxicity. The colony-forming efficiencies (CFE) of these cells were determined using a lung colony assay. Synchronized cell population of FSa cells, separated by centrifugal elutriation, were injected into recipient animals and exposed 20min later to a single dose of VCR to determine their age-specific sensitivity. Under these conditions there appeared to be a suggestion of an enhanced killing of cells enriched in the G2 + M phase. However, following prolonged VCR exposure in vitro (e.g., 2.5pgml-1; 25ml for 24h) to primary cultures of FSa cells or in vivo (e.g., 0.25mgkg-1 per fraction i.p.; 5 fractions in 24h) to macroscopic pulmonary tumour nodules, elutriated FSa cell populations most enriched with G1 phase cells exhibited the lowest CFE. If under either condition exposed cells were allowed to recover in the absence of VCR for 24h prior to their removal and separation, FSa cell survival increased in each of the elutriated populations. In contrast, while G1 enriched cell populations from in vitro exposed cultures still exhibited a significant reduction in CFE, no such age-specific response was observed for in vivo exposed macroscopic pulmonary nodules. The stathomokinetic effect of VCR on FSa cells was also readily observed in vitro using FMF analysis (e.g., an increase of from 20 to 38% in the G2+M phase compartment). While no such effect was observed in vivo using FMF analysis, cluster of mitotic figures were observed. The mitotic indices (MI) of the in vivo exposed FSa cells increased from 2.5 to 8.4%.
British Journal of Cancer, Mar 1, 1982
The cytotoxic effects in vivo of hydroxyurea (HU) on murine fibrosarcoma (FSa) cells grown as pul... more The cytotoxic effects in vivo of hydroxyurea (HU) on murine fibrosarcoma (FSa) cells grown as pulmonary tumours were determined. Tumour cells from 13-day-old nodules were made into suspension and separated on the basis of cell size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations, as well as the degree of contamination by normal diploid cells in each of the tumour-cell populations. HU cytotoxicity was tested by administering both a single 1 mg/g i.p. dose into mice that had been injected i.v. 20 min earlier with known numbers of synchronized viable FSa cells, and i.p. doses of 1 mg/g each into mice bearing 13-day-old pulmonary nodules. In the latter experiments, animals were killed 1 h after the last dose, and the tumour nodules were excised and made into a single-cell suspension and elutriated. Known numbers of cells from each fraction were injected into recipient mice to determine survival. In both sets of experiments, cell killing by HU correlated with the percentage of S-phase cells. The treatment of 13-day-old pulmonary nodules with 3 doses of HU also depleted the (G2+M) phase tumour cells and increased the heterogeneity between tumour subpopulations, as determined by FMF analysis.
University of North Texas Digital Library (University of North Texas), 1990
The submitted manuscript has been authored by a contractor of the U. S. Government under contract... more The submitted manuscript has been authored by a contractor of the U. S. Government under contract No. W-31-109-ENG-2 3. Accordingly, the US. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do », for U. S. Government purposes.
Radiation Research, 2017
One of the most concerning side effects of exposure to radiation are the carcinogenic risks. To r... more One of the most concerning side effects of exposure to radiation are the carcinogenic risks. To reduce the negative effects of radiation, both cytoprotective and radioprotective agents have been developed. However, little is known regarding their potential for suppressing carcinogenesis. Andrographis paniculata, a plant, with multiple medicinal uses that is commonly used in traditional medicine, has three major constituents known to have cellular antioxidant activity: andrographolide (AP1); 14-deoxy-11,12-didehydroandrographolide (AP3); and neoandrographolide (AP4). In our study, we tested these elements for their radioprotective properties as well as their anti-neoplastic effects on transformation using the BALB/3T3 cell model. All three compounds were able to reduce radiation-induced DNA damage. However, AP4 appeared to have superior radioprotective properties compared to the other two compounds, presumably by protecting mitochondrial function. The compound was able to suppress radiation-induced cellular transformation through inhibition of STAT3. Treatment with AP4 also reduced expressions of MMP-2 and MMP-9. These results suggest that AP4 could be further studied and developed into an anti-transformation/carcinogenic drug as well as a radioprotective agent.
European Journal of Cancer and Clinical Oncology, 1987
Environmental and Molecular Mutagenesis, 1995
The role of DNA double-strand breaks in producing mitotic (Gâ) delay was examined in Chinese hams... more The role of DNA double-strand breaks in producing mitotic (Gâ) delay was examined in Chinese hamster ovary (CHO) cells. Restriction enzymes, which produce only DNA double-stranded breaks, were introduced by electroporation into CHO-K1 and its radiation-sensitive derivativexrs-5; at several time points after treatment, the fraction of cells in Gâ was determined. Electroporation of Alu I or Sau 3A1 into CHO-K1
Radiation Research, 1988
The radioprotector 2-[aminopropyl)amino] ethanethiol (WR1065), which has been reported to reduce ... more The radioprotector 2-[aminopropyl)amino] ethanethiol (WR1065), which has been reported to reduce the cytotoxic and mutagenic effects of low LET radiation, was investigated for its ability to protect against low LET (60Co gamma ray) and high LET (fission-spectrum neutron)-induced chromosome damage in V79 cells. Cells were irradiated in G2 phase in the presence or absence of 4 mM WR1065 and were harvested and analyzed 2 h later for chromatid-type aberrations. Irradiation of G2-phase V79 cells in the presence of WR1065 resulted in a 30 to 50% reduction in the frequency of gamma-ray and neutron-induced chromatid-type breaks and exchanges. The effects were found only after exposures of greater than 200 cGy gamma-ray or 50 cGy neutron irradiation. The radioprotector was effective at reducing neutron-induced aberrations after exposures at dose rates of both 10 and 43 cGy/min. Thus the radioprotector WR1065 is an effective anti-clastogenic agent in V79 cells, protecting against both 60Co gamma-ray and fission-spectrum neutron-induced aberrations, when present during irradiation.
Radiation Research, 1990
The systemic effects of the radiation protective agent, S-3-(3-methylaminopropylamino) propylphos... more The systemic effects of the radiation protective agent, S-3-(3-methylaminopropylamino) propylphosphorothioic acid (WR-151327), were studied in unirradiated B6CF1 male mice. Fifty mice were injected intraperitoneally with 540 mg/kg WR-151327, and groups of five mice were sacrificed at 14-day intervals up to and including 140 days post-treatment. Ten mice served as sham-injected controls. A necropsy was performed and gross morphological abnormalities were noted. Tissues (brain, eyes, harderian gland, salivary glands, sternal bone marrow, thyroid, lung, thymus, esophagus, trachea, skeletal muscle, heart, liver, kidney, adrenal gland, spleen, small intestine, pancreas, and testes) were fixed in 10% formalin, embedded in paraffin, and sectioned. Slides were routinely stained with hematoxylin and eosin while Alizarin red stain was used to test specifically for the presence of calcium salts. Histopathological effects of WR-151327 were restricted to the testes, salivary gland, and pancreas. The caudal pole of the testes was observed to undergo progressive changes from coagulation necrosis to dystrophic calcification. The cells of the submandibular salivary gland showed mainly hyperchromatic nuclei while the pancreas showed enlarged islets of Langerhans.
Pharmacology & Therapeutics, 1988
Mutagenesis, 1994
The polyamine spermine and the disulfide N,N"-(dithiodi-2,1-ethanediyl)bis-1,3-propanedi... more The polyamine spermine and the disulfide N,N"-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278) are structurally similar agents capable of binding to DNA. WR-33278 is the disulfide moiety of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Because of their reported structural and functional similarities, it was of interest to compare their effects on cell survival and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in Chinese hamster AA8 cells. WR-33278 and spermine (at concentrations of 0.01 and 0.001 mM) were electroporated into cells. Electroporation, 300 V and 125 microF, was performed either 30 min prior to or 3 h following exposure of cells to 750 cGy of ionizing radiation. Electroporation alone reduced cell survival to 75% but had no effect on hprt mutation frequency. The electroporation of either spermine or WR-33278 at concentrations greater than 0.01 mM was extremely toxic. The exposure of cells to both electroporation and irradiation gave rise to enhanced cell killing and mutation induction, with the sequence of irradiation followed 3 h later by electroporation being the more toxic protocol. Cell survival was only enhanced following electroporation of 0.01 mM of spermine and WR-33278 30 min prior to irradiation. Protection against radiation-induced hprt mutations was observed for both spermine and WR-33278 under all experimental conditions tested. Spermine at exposure concentrations of 0.01 and 0.001 mM administered 30 min before or 3 h after irradiation reduced mutation frequencies by factors of 2.2, 1.2, 1.9 and 2.2, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
International Journal of Radiation Oncology*Biology*Physics, 1984
The survival of cells from two murine fibrosarcoma (Fsa) subpopulrtions after exposure to radiati... more The survival of cells from two murine fibrosarcoma (Fsa) subpopulrtions after exposure to radiation only or radiation in combination with misonidazole (0.2 mg/g/fraction) was determined using a lung colony assay. FSa tumors grown in the hind legs of C,Hf/Kam pathogen-free mice were irradiated in situ when the tumors were 8 to 10 mm in diameter. Single cell suspensions prepared from excised tumors were separated on linear deusity gradients of Renografin, and the clonogenicity of predominantly oxic Band 2 (density 1.08 g/cm3) and predominantly hypoxic Band 4 (density 1.14 g/cm') cells were measured. The surviving fraction of cells after dosea of 1,2, and 3 Gy, alone or preceded 30 minutea earlier with an i.p. injection of misonidazole (0.2 mg/g) was estimated from that measured after total radiation doses of 5 Gy = 5 x 1 Gy, 10 Gy = 5 x 2 Gy, and 15 Gy = 5 x 3 Gy, with the misonidazoletreated groups receiving a total drug dose of 1 mg/g, under the assumption of an equal effect per fraction. Under these conditions the initial slopes of Band 2 cells following irradiation only or irradiation plus misonidaxole were ,D@ = 3.6 Gy and 2.74 Gy, respectively, giving rise to a sensitizer enhancement ratio of 1.3. Band 4 cells exhibited a ,Do of 5.15 Gy to radiation only and 2.75 Gy to radiation plus misonidazole (SER of 1.9). In addition, misonidazole when administered alone in a single dose or up to 5 fractious of 0.2 mg/g each separated by 4-hour intervals, was effective in killing 50% of the Band 4 cells. The target population at risk appeared to remain constant regardless of the number of dose fractions administered. In contrast, Band 2 cells were not affected by the cytotoxic action of misonidazole. These data suggest that misonidazole is effective in sensitizing hypoxic cells in the clinical dose range, and that it is directly cytotoxic to hypoxic tumor cells. Misonidazole, Equal effect per fraction, Low dose, in silu, Tumor.
International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine, 1987
Both S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) and 16-16 dimethyl prostaglandi... more Both S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) and 16-16 dimethyl prostaglandin E2 (dm PGE2) protected the intestinal clonogenic cells to some degree from the effects of 137Cs gamma-irradiation. The D0 was increased from 1.1 +/- 0.12 Gy in controls to 1.55 +/- 0.48 Gy in 16-16 dm PGE2 treated and 2.12 +/- 0.20 Gy in WR-2721 treated mice. Both agents also increased the shoulder of the clonogenic-cell survival curve. Studies were done to measure the effects of these two different radioprotectors on radiation-induction of DNA single-strand breaks in cells comprising the murine intestinal mucosa. The number of DNA single-strand breaks increased with increasing doses of gamma-rays in animals killed immediately following exposure. WR-2721 reduced the number of initial radiation-induced DNA single-strand breaks when given one-half hour before exposure; the time of maximum protection. In contrast, 16-16 dm PGE2 given 1 hour before irradiation (the time required to afford maximum protection from radiation cytotoxicity) did not reduce the number of initial DNA breaks. Both agents impeded the rate of rejoining of DNA breaks with increasing time after irradiation. However, the relationship between these effects on the rate of strand rejoining and cell survival is unknown. These results suggest that either both agents are similarly distributed within the cells but the mechanisms of radioprotection are different, or the mechanisms by which these agents protect are similar, but the two agents affect different subcellular targets, the protection of which contributes to increased cell survival.
Drug Metabolism Reviews, 1989
Clinical & Experimental Metastasis, 1989
Tumor cells from a murine fibrosarcoma (FSa) produce plasminogen activator (PA), a protease that ... more Tumor cells from a murine fibrosarcoma (FSa) produce plasminogen activator (PA), a protease that converts the zymogen plasminogen into the trypsin-like enzyme plasmin. Several studies indicate that tumor cell invasion is accompanied by proteolysis and that PA, generated by highly malignant cells, is by far the most ubiquitous protease associated with malignant transformation. Subpopulations of FSa cells were isolated by using density gradient centrifugation and the ability of these populations to form lung colonies was compared with their associated levels of PA production. Five populations of cells from a murine fibrosarcoma were separated in continuous gradients of Renografin in the density range 1.05-1.18 g/cm2. The PA activities of an unseparated control cell lysate and cell lysates of the five separated populations were determined by using [125I]fibrin as a substrate in a reaction between cell lysate and plasminogen. The assay was based on the release of digested [125I]fibrin from the surface of Petri dishes into the supernatant solution, and the results were expressed as a percentage of the total radioactivity. The cell populations collected at densities of 1.05 and 1.09 (B1, B2) were the more clonogenic with relative clonogenic efficiencies of 2.6 and 3.3 times that of the unseparated tumor population, respectively. Analysis for PA demonstrated that enzyme formation was restricted mostly to these two populations. Cells from populations 4 and 5 did not secrete increased amounts of PA and had reduced clonogenic efficiencies compared with the unseparated FSa control population. These results are consistent with the hypothesis that PA activity is correlated with the clonogenicity of tumor subpopulations isolated from a heterogeneous and complex tumor system such as the FSa.
Carcinogenesis, 1994
The biological effects of exposures to high LET radiations have particular relevance to radiation... more The biological effects of exposures to high LET radiations have particular relevance to radiation protection and risk assessment. Since most cancers are of epithelial origin, it is important to obtain a better understanding of radiation-induced oncogenic transformation in this cell type. Accordingly we have initiated studies to determine whether immortalized human epidermal keratinocytes (RHEK) can be transformed with high LET radiations. Exponentially growing RHEK cells were treated with single doses (1, 10, 25, 50 and 100 cGy) of 0.85 MeV fission neutrons from the Janus reactor. Neutron exposure led to the development of morphologically altered cells and foci formation after 6 weeks at confluence. These transformed cultures grew with an increased saturation density, exhibited anchorage-independent growth and formed tumors in athymic mice. Single-strand conformational polymorphism analysis and DNA sequencing demonstrated the absence of point mutations in codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genes and exons 4-9 of the p53 tumor suppressor gene. These studies demonstrate that high LET radiations (fission neutrons) can transform immortalized human epithelial cells to a malignant phenotype that does not appear to involve mutations in either the cellular p53 or ras genes.
Carcinogenesis, 1986
To investigate mechanisms underlying the formation of carcinogen-induced altered hepatocyte foci,... more To investigate mechanisms underlying the formation of carcinogen-induced altered hepatocyte foci, we histochemically examined the livers of 150-day-old rats that had been treated neonatally with single doses of gamma radiation (75, 150 or 300 rad, whole body) and i.p.-injected diethylnitrosamine (DEN, 0.15 mumol/g body wt) either separately or in combination. Three focus phenotypes, showing the elevated gamma-glutamyl transpeptidase [GG(+)] and/or the iron-exclusion [Fe(-)] histochemical marker(s) were quantitated through the use of serial frozen sectioning techniques and computer-assisted image analysis. DEN and gamma radiation each induced foci when given separately but total focus yield per cm3 of liver was more than 10-fold greater in DEN-treated than in irradiated rats and approximately 3-fold higher in females than in males. Combining the DEN and radiation treatments synergistically increased total focus yields for both sexes, although this response declined with increasing radiation dosage. For rats receiving DEN alone, the sex-dependent differential in total focus yield was due to the higher frequencies in females of the iron-excluding phenotypes [Fe(-) alone and Fe(-) + GG(+)]; the frequencies of foci showing only GG(+) were similar in both sexes. In contrast, the enhancement in total focus yield resulting from the combined DEN--radiation treatments was primarily a consequence of increases in the foci with GG(+) alone. The results suggest that (a) qualitatively different types of genetic damage (carcinogen-induced point mutations and radiation-induced rearrangements) may interact synergistically in the induction of phenotypically altered cells and (b) separate genetic loci are involved in the sex-mediated modulation of focus production and the synergistic enhancement of focus production by DEN--gamma radiation interactions.
Carcinogenesis, 1987
To characterize the effects of combined treatments with gamma radiation and diethylnitrosamine (D... more To characterize the effects of combined treatments with gamma radiation and diethylnitrosamine (DEN) on the induction of histochemically detectable altered hepatocyte foci and hepatic tumors, we assessed the yields of these lesions in the livers of 150-day-old rats that had been treated neonatally with a single dose of gamma radiation (75 rad, whole body) and i.p.-injected DEN (0.15 mumol/g body wt), either separately or in combination. The combined treatments involved the administration of the two stimuli in both possible sequences, with the interval between treatments set at 1 h. The focus population was examined for two histochemical markers (elevated gamma-glutamyl transpeptidase [GGT(+)] and iron exclusion [FE(-)], giving rise to three detectable focus phenotypes, i.e. GGT(+) foci, FE(-) foci, and GGT(+), FE(-) foci. Frequencies of the three phenotypes were quantitated through the use of serial frozen sectioning techniques and computer-assisted image analysis. GGT(+) focus induction was synergistically enhanced by the combined treatment irrespective of the order in which the two stimuli were administered; the remaining two phenotypes did not show such enhancement. The magnitude of the GGT(+) focus response was significantly greater when the treatment sequence was gamma----DEN as opposed to DEN----gamma. Tumor yields in rats receiving combined gamma--DEN treatment were similar to those in rats receiving the DEN alone, irrespective of the gamma--DEN treatment sequence. These results suggest that phenotypically distinguishable lesions, including foci with different histochemical marker patterns and tumors, originate from specific types of damage at different genetic loci and are developmentally independent; and the expression of the GGT(+) marker per se in altered hepatocyte foci is not a reliable index of incipient hepatic neoplasia.
Carcinogenesis, 1986
We have studied the effect of 2-[(aminopropyl)amino]ethanethiol (WR1065) on the induction of neop... more We have studied the effect of 2-[(aminopropyl)amino]ethanethiol (WR1065) on the induction of neoplastic transformation using 10T1/2 cells and on mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus using Chinese hamster V79 cells. Here we report the first observations that treatment of 10T1/2 cells with 1 mM WR1065 for a total of 35 min during irradiation with 60Co gamma-rays significantly reduces the incidence of neoplastic transformation while having no effect on cell viability. In a similar experiment with V79 cells in which 4 mM WR1065 was used, we found a significant reduction in mutation frequency at the HGPRT locus and significant protection against cell killing. These results suggest that WR1065 acts to modulate both acute damage and sub-lethal processes that lead to mutation and neoplastic transformation. Beyond the purely mechanistic approach of these studies, the potential application of these agents to minimizing the long-term neoplastic effects of radiation or chemotherapeutic agents currently in use for treating potentially curable cancer patients should be further investigated.
American Journal of Clinical Oncology, 1987
Sixty patients with high-grade gliomas (including 50 patients with glioblastoma multiforme) were ... more Sixty patients with high-grade gliomas (including 50 patients with glioblastoma multiforme) were entered on four sequential Phase I trials combining continuous intravenous infusions of halogenated pyrimidines and high-dose brain irradiation. Patients received two 14-day infusions of bromodeoxyuridine (BUdR) or iododeoxyuridine (IUdR) during the initial wide field and later reduced field radiation treatment (total radiation dose 65-70 Gy). All patients were
Cancer Research, 2018
Circadian clocks are intimately involved in the homeostatic maintenance of metabolic and physiolo... more Circadian clocks are intimately involved in the homeostatic maintenance of metabolic and physiological processes that may have increasingly important roles to enhance bone marrow transplantation success in cancer patients; to protect normal tissue surrounding tumors from the harmful effects of radio/chemotherapy; and to enhance the quality of diet and sleep for astronauts during space exploration. Herein, we report that expression of PERIOD2 (PER2; a core circadian clock component and “the inherent driver” of radioprotection) is required for adaptive protection against environmental radiation stress. PER2 expression is induced by exposure to low dose radiation (LDR; 10cGy) and siRNA blockade of PER2 ablates LDR-induced adaptive radioprotection. In addition, melatonin (N-acetyl-5-methoxytryptamine), a natural anti-oxidant and hypothalamic circadian synchronizer, stimulates PER2 expression and confers mice radioprotection through weight maintenance and increased survival. LDR-induced ...
British Journal of Cancer, Aug 1, 1983
The cytotoxic and stathomokinetic effects of vincristine (VCR) on murine fibrosarcoma (FSa) cells... more The cytotoxic and stathomokinetic effects of vincristine (VCR) on murine fibrosarcoma (FSa) cells, grown either in vitro as primary cultures or in vivo as microor macroscopic pulmonary nodules, were determined and compared. FSa cells were separated and synchronized on the basis of size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations, thus allowing determination of age-dependent cytotoxicity. The colony-forming efficiencies (CFE) of these cells were determined using a lung colony assay. Synchronized cell population of FSa cells, separated by centrifugal elutriation, were injected into recipient animals and exposed 20min later to a single dose of VCR to determine their age-specific sensitivity. Under these conditions there appeared to be a suggestion of an enhanced killing of cells enriched in the G2 + M phase. However, following prolonged VCR exposure in vitro (e.g., 2.5pgml-1; 25ml for 24h) to primary cultures of FSa cells or in vivo (e.g., 0.25mgkg-1 per fraction i.p.; 5 fractions in 24h) to macroscopic pulmonary tumour nodules, elutriated FSa cell populations most enriched with G1 phase cells exhibited the lowest CFE. If under either condition exposed cells were allowed to recover in the absence of VCR for 24h prior to their removal and separation, FSa cell survival increased in each of the elutriated populations. In contrast, while G1 enriched cell populations from in vitro exposed cultures still exhibited a significant reduction in CFE, no such age-specific response was observed for in vivo exposed macroscopic pulmonary nodules. The stathomokinetic effect of VCR on FSa cells was also readily observed in vitro using FMF analysis (e.g., an increase of from 20 to 38% in the G2+M phase compartment). While no such effect was observed in vivo using FMF analysis, cluster of mitotic figures were observed. The mitotic indices (MI) of the in vivo exposed FSa cells increased from 2.5 to 8.4%.
British Journal of Cancer, Mar 1, 1982
The cytotoxic effects in vivo of hydroxyurea (HU) on murine fibrosarcoma (FSa) cells grown as pul... more The cytotoxic effects in vivo of hydroxyurea (HU) on murine fibrosarcoma (FSa) cells grown as pulmonary tumours were determined. Tumour cells from 13-day-old nodules were made into suspension and separated on the basis of cell size by centrifugal elutriation. Flow microfluorometry (FMF) was used to determine the cell-cycle parameters and the relative synchrony of the separated populations, as well as the degree of contamination by normal diploid cells in each of the tumour-cell populations. HU cytotoxicity was tested by administering both a single 1 mg/g i.p. dose into mice that had been injected i.v. 20 min earlier with known numbers of synchronized viable FSa cells, and i.p. doses of 1 mg/g each into mice bearing 13-day-old pulmonary nodules. In the latter experiments, animals were killed 1 h after the last dose, and the tumour nodules were excised and made into a single-cell suspension and elutriated. Known numbers of cells from each fraction were injected into recipient mice to determine survival. In both sets of experiments, cell killing by HU correlated with the percentage of S-phase cells. The treatment of 13-day-old pulmonary nodules with 3 doses of HU also depleted the (G2+M) phase tumour cells and increased the heterogeneity between tumour subpopulations, as determined by FMF analysis.
University of North Texas Digital Library (University of North Texas), 1990
The submitted manuscript has been authored by a contractor of the U. S. Government under contract... more The submitted manuscript has been authored by a contractor of the U. S. Government under contract No. W-31-109-ENG-2 3. Accordingly, the US. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do », for U. S. Government purposes.
Radiation Research, 2017
One of the most concerning side effects of exposure to radiation are the carcinogenic risks. To r... more One of the most concerning side effects of exposure to radiation are the carcinogenic risks. To reduce the negative effects of radiation, both cytoprotective and radioprotective agents have been developed. However, little is known regarding their potential for suppressing carcinogenesis. Andrographis paniculata, a plant, with multiple medicinal uses that is commonly used in traditional medicine, has three major constituents known to have cellular antioxidant activity: andrographolide (AP1); 14-deoxy-11,12-didehydroandrographolide (AP3); and neoandrographolide (AP4). In our study, we tested these elements for their radioprotective properties as well as their anti-neoplastic effects on transformation using the BALB/3T3 cell model. All three compounds were able to reduce radiation-induced DNA damage. However, AP4 appeared to have superior radioprotective properties compared to the other two compounds, presumably by protecting mitochondrial function. The compound was able to suppress radiation-induced cellular transformation through inhibition of STAT3. Treatment with AP4 also reduced expressions of MMP-2 and MMP-9. These results suggest that AP4 could be further studied and developed into an anti-transformation/carcinogenic drug as well as a radioprotective agent.
European Journal of Cancer and Clinical Oncology, 1987
Environmental and Molecular Mutagenesis, 1995
The role of DNA double-strand breaks in producing mitotic (Gâ) delay was examined in Chinese hams... more The role of DNA double-strand breaks in producing mitotic (Gâ) delay was examined in Chinese hamster ovary (CHO) cells. Restriction enzymes, which produce only DNA double-stranded breaks, were introduced by electroporation into CHO-K1 and its radiation-sensitive derivativexrs-5; at several time points after treatment, the fraction of cells in Gâ was determined. Electroporation of Alu I or Sau 3A1 into CHO-K1
Radiation Research, 1988
The radioprotector 2-[aminopropyl)amino] ethanethiol (WR1065), which has been reported to reduce ... more The radioprotector 2-[aminopropyl)amino] ethanethiol (WR1065), which has been reported to reduce the cytotoxic and mutagenic effects of low LET radiation, was investigated for its ability to protect against low LET (60Co gamma ray) and high LET (fission-spectrum neutron)-induced chromosome damage in V79 cells. Cells were irradiated in G2 phase in the presence or absence of 4 mM WR1065 and were harvested and analyzed 2 h later for chromatid-type aberrations. Irradiation of G2-phase V79 cells in the presence of WR1065 resulted in a 30 to 50% reduction in the frequency of gamma-ray and neutron-induced chromatid-type breaks and exchanges. The effects were found only after exposures of greater than 200 cGy gamma-ray or 50 cGy neutron irradiation. The radioprotector was effective at reducing neutron-induced aberrations after exposures at dose rates of both 10 and 43 cGy/min. Thus the radioprotector WR1065 is an effective anti-clastogenic agent in V79 cells, protecting against both 60Co gamma-ray and fission-spectrum neutron-induced aberrations, when present during irradiation.
Radiation Research, 1990
The systemic effects of the radiation protective agent, S-3-(3-methylaminopropylamino) propylphos... more The systemic effects of the radiation protective agent, S-3-(3-methylaminopropylamino) propylphosphorothioic acid (WR-151327), were studied in unirradiated B6CF1 male mice. Fifty mice were injected intraperitoneally with 540 mg/kg WR-151327, and groups of five mice were sacrificed at 14-day intervals up to and including 140 days post-treatment. Ten mice served as sham-injected controls. A necropsy was performed and gross morphological abnormalities were noted. Tissues (brain, eyes, harderian gland, salivary glands, sternal bone marrow, thyroid, lung, thymus, esophagus, trachea, skeletal muscle, heart, liver, kidney, adrenal gland, spleen, small intestine, pancreas, and testes) were fixed in 10% formalin, embedded in paraffin, and sectioned. Slides were routinely stained with hematoxylin and eosin while Alizarin red stain was used to test specifically for the presence of calcium salts. Histopathological effects of WR-151327 were restricted to the testes, salivary gland, and pancreas. The caudal pole of the testes was observed to undergo progressive changes from coagulation necrosis to dystrophic calcification. The cells of the submandibular salivary gland showed mainly hyperchromatic nuclei while the pancreas showed enlarged islets of Langerhans.
Pharmacology & Therapeutics, 1988
Mutagenesis, 1994
The polyamine spermine and the disulfide N,N"-(dithiodi-2,1-ethanediyl)bis-1,3-propanedi... more The polyamine spermine and the disulfide N,N"-(dithiodi-2,1-ethanediyl)bis-1,3-propanediamine (WR-33278) are structurally similar agents capable of binding to DNA. WR-33278 is the disulfide moiety of the clinically studied radioprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721). Because of their reported structural and functional similarities, it was of interest to compare their effects on cell survival and mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in Chinese hamster AA8 cells. WR-33278 and spermine (at concentrations of 0.01 and 0.001 mM) were electroporated into cells. Electroporation, 300 V and 125 microF, was performed either 30 min prior to or 3 h following exposure of cells to 750 cGy of ionizing radiation. Electroporation alone reduced cell survival to 75% but had no effect on hprt mutation frequency. The electroporation of either spermine or WR-33278 at concentrations greater than 0.01 mM was extremely toxic. The exposure of cells to both electroporation and irradiation gave rise to enhanced cell killing and mutation induction, with the sequence of irradiation followed 3 h later by electroporation being the more toxic protocol. Cell survival was only enhanced following electroporation of 0.01 mM of spermine and WR-33278 30 min prior to irradiation. Protection against radiation-induced hprt mutations was observed for both spermine and WR-33278 under all experimental conditions tested. Spermine at exposure concentrations of 0.01 and 0.001 mM administered 30 min before or 3 h after irradiation reduced mutation frequencies by factors of 2.2, 1.2, 1.9 and 2.2, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
International Journal of Radiation Oncology*Biology*Physics, 1984
The survival of cells from two murine fibrosarcoma (Fsa) subpopulrtions after exposure to radiati... more The survival of cells from two murine fibrosarcoma (Fsa) subpopulrtions after exposure to radiation only or radiation in combination with misonidazole (0.2 mg/g/fraction) was determined using a lung colony assay. FSa tumors grown in the hind legs of C,Hf/Kam pathogen-free mice were irradiated in situ when the tumors were 8 to 10 mm in diameter. Single cell suspensions prepared from excised tumors were separated on linear deusity gradients of Renografin, and the clonogenicity of predominantly oxic Band 2 (density 1.08 g/cm3) and predominantly hypoxic Band 4 (density 1.14 g/cm') cells were measured. The surviving fraction of cells after dosea of 1,2, and 3 Gy, alone or preceded 30 minutea earlier with an i.p. injection of misonidazole (0.2 mg/g) was estimated from that measured after total radiation doses of 5 Gy = 5 x 1 Gy, 10 Gy = 5 x 2 Gy, and 15 Gy = 5 x 3 Gy, with the misonidazoletreated groups receiving a total drug dose of 1 mg/g, under the assumption of an equal effect per fraction. Under these conditions the initial slopes of Band 2 cells following irradiation only or irradiation plus misonidaxole were ,D@ = 3.6 Gy and 2.74 Gy, respectively, giving rise to a sensitizer enhancement ratio of 1.3. Band 4 cells exhibited a ,Do of 5.15 Gy to radiation only and 2.75 Gy to radiation plus misonidazole (SER of 1.9). In addition, misonidazole when administered alone in a single dose or up to 5 fractious of 0.2 mg/g each separated by 4-hour intervals, was effective in killing 50% of the Band 4 cells. The target population at risk appeared to remain constant regardless of the number of dose fractions administered. In contrast, Band 2 cells were not affected by the cytotoxic action of misonidazole. These data suggest that misonidazole is effective in sensitizing hypoxic cells in the clinical dose range, and that it is directly cytotoxic to hypoxic tumor cells. Misonidazole, Equal effect per fraction, Low dose, in silu, Tumor.
International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine, 1987
Both S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) and 16-16 dimethyl prostaglandi... more Both S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) and 16-16 dimethyl prostaglandin E2 (dm PGE2) protected the intestinal clonogenic cells to some degree from the effects of 137Cs gamma-irradiation. The D0 was increased from 1.1 +/- 0.12 Gy in controls to 1.55 +/- 0.48 Gy in 16-16 dm PGE2 treated and 2.12 +/- 0.20 Gy in WR-2721 treated mice. Both agents also increased the shoulder of the clonogenic-cell survival curve. Studies were done to measure the effects of these two different radioprotectors on radiation-induction of DNA single-strand breaks in cells comprising the murine intestinal mucosa. The number of DNA single-strand breaks increased with increasing doses of gamma-rays in animals killed immediately following exposure. WR-2721 reduced the number of initial radiation-induced DNA single-strand breaks when given one-half hour before exposure; the time of maximum protection. In contrast, 16-16 dm PGE2 given 1 hour before irradiation (the time required to afford maximum protection from radiation cytotoxicity) did not reduce the number of initial DNA breaks. Both agents impeded the rate of rejoining of DNA breaks with increasing time after irradiation. However, the relationship between these effects on the rate of strand rejoining and cell survival is unknown. These results suggest that either both agents are similarly distributed within the cells but the mechanisms of radioprotection are different, or the mechanisms by which these agents protect are similar, but the two agents affect different subcellular targets, the protection of which contributes to increased cell survival.
Drug Metabolism Reviews, 1989
Clinical & Experimental Metastasis, 1989
Tumor cells from a murine fibrosarcoma (FSa) produce plasminogen activator (PA), a protease that ... more Tumor cells from a murine fibrosarcoma (FSa) produce plasminogen activator (PA), a protease that converts the zymogen plasminogen into the trypsin-like enzyme plasmin. Several studies indicate that tumor cell invasion is accompanied by proteolysis and that PA, generated by highly malignant cells, is by far the most ubiquitous protease associated with malignant transformation. Subpopulations of FSa cells were isolated by using density gradient centrifugation and the ability of these populations to form lung colonies was compared with their associated levels of PA production. Five populations of cells from a murine fibrosarcoma were separated in continuous gradients of Renografin in the density range 1.05-1.18 g/cm2. The PA activities of an unseparated control cell lysate and cell lysates of the five separated populations were determined by using [125I]fibrin as a substrate in a reaction between cell lysate and plasminogen. The assay was based on the release of digested [125I]fibrin from the surface of Petri dishes into the supernatant solution, and the results were expressed as a percentage of the total radioactivity. The cell populations collected at densities of 1.05 and 1.09 (B1, B2) were the more clonogenic with relative clonogenic efficiencies of 2.6 and 3.3 times that of the unseparated tumor population, respectively. Analysis for PA demonstrated that enzyme formation was restricted mostly to these two populations. Cells from populations 4 and 5 did not secrete increased amounts of PA and had reduced clonogenic efficiencies compared with the unseparated FSa control population. These results are consistent with the hypothesis that PA activity is correlated with the clonogenicity of tumor subpopulations isolated from a heterogeneous and complex tumor system such as the FSa.
Carcinogenesis, 1994
The biological effects of exposures to high LET radiations have particular relevance to radiation... more The biological effects of exposures to high LET radiations have particular relevance to radiation protection and risk assessment. Since most cancers are of epithelial origin, it is important to obtain a better understanding of radiation-induced oncogenic transformation in this cell type. Accordingly we have initiated studies to determine whether immortalized human epidermal keratinocytes (RHEK) can be transformed with high LET radiations. Exponentially growing RHEK cells were treated with single doses (1, 10, 25, 50 and 100 cGy) of 0.85 MeV fission neutrons from the Janus reactor. Neutron exposure led to the development of morphologically altered cells and foci formation after 6 weeks at confluence. These transformed cultures grew with an increased saturation density, exhibited anchorage-independent growth and formed tumors in athymic mice. Single-strand conformational polymorphism analysis and DNA sequencing demonstrated the absence of point mutations in codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genes and exons 4-9 of the p53 tumor suppressor gene. These studies demonstrate that high LET radiations (fission neutrons) can transform immortalized human epithelial cells to a malignant phenotype that does not appear to involve mutations in either the cellular p53 or ras genes.
Carcinogenesis, 1986
To investigate mechanisms underlying the formation of carcinogen-induced altered hepatocyte foci,... more To investigate mechanisms underlying the formation of carcinogen-induced altered hepatocyte foci, we histochemically examined the livers of 150-day-old rats that had been treated neonatally with single doses of gamma radiation (75, 150 or 300 rad, whole body) and i.p.-injected diethylnitrosamine (DEN, 0.15 mumol/g body wt) either separately or in combination. Three focus phenotypes, showing the elevated gamma-glutamyl transpeptidase [GG(+)] and/or the iron-exclusion [Fe(-)] histochemical marker(s) were quantitated through the use of serial frozen sectioning techniques and computer-assisted image analysis. DEN and gamma radiation each induced foci when given separately but total focus yield per cm3 of liver was more than 10-fold greater in DEN-treated than in irradiated rats and approximately 3-fold higher in females than in males. Combining the DEN and radiation treatments synergistically increased total focus yields for both sexes, although this response declined with increasing radiation dosage. For rats receiving DEN alone, the sex-dependent differential in total focus yield was due to the higher frequencies in females of the iron-excluding phenotypes [Fe(-) alone and Fe(-) + GG(+)]; the frequencies of foci showing only GG(+) were similar in both sexes. In contrast, the enhancement in total focus yield resulting from the combined DEN--radiation treatments was primarily a consequence of increases in the foci with GG(+) alone. The results suggest that (a) qualitatively different types of genetic damage (carcinogen-induced point mutations and radiation-induced rearrangements) may interact synergistically in the induction of phenotypically altered cells and (b) separate genetic loci are involved in the sex-mediated modulation of focus production and the synergistic enhancement of focus production by DEN--gamma radiation interactions.
Carcinogenesis, 1987
To characterize the effects of combined treatments with gamma radiation and diethylnitrosamine (D... more To characterize the effects of combined treatments with gamma radiation and diethylnitrosamine (DEN) on the induction of histochemically detectable altered hepatocyte foci and hepatic tumors, we assessed the yields of these lesions in the livers of 150-day-old rats that had been treated neonatally with a single dose of gamma radiation (75 rad, whole body) and i.p.-injected DEN (0.15 mumol/g body wt), either separately or in combination. The combined treatments involved the administration of the two stimuli in both possible sequences, with the interval between treatments set at 1 h. The focus population was examined for two histochemical markers (elevated gamma-glutamyl transpeptidase [GGT(+)] and iron exclusion [FE(-)], giving rise to three detectable focus phenotypes, i.e. GGT(+) foci, FE(-) foci, and GGT(+), FE(-) foci. Frequencies of the three phenotypes were quantitated through the use of serial frozen sectioning techniques and computer-assisted image analysis. GGT(+) focus induction was synergistically enhanced by the combined treatment irrespective of the order in which the two stimuli were administered; the remaining two phenotypes did not show such enhancement. The magnitude of the GGT(+) focus response was significantly greater when the treatment sequence was gamma----DEN as opposed to DEN----gamma. Tumor yields in rats receiving combined gamma--DEN treatment were similar to those in rats receiving the DEN alone, irrespective of the gamma--DEN treatment sequence. These results suggest that phenotypically distinguishable lesions, including foci with different histochemical marker patterns and tumors, originate from specific types of damage at different genetic loci and are developmentally independent; and the expression of the GGT(+) marker per se in altered hepatocyte foci is not a reliable index of incipient hepatic neoplasia.
Carcinogenesis, 1986
We have studied the effect of 2-[(aminopropyl)amino]ethanethiol (WR1065) on the induction of neop... more We have studied the effect of 2-[(aminopropyl)amino]ethanethiol (WR1065) on the induction of neoplastic transformation using 10T1/2 cells and on mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus using Chinese hamster V79 cells. Here we report the first observations that treatment of 10T1/2 cells with 1 mM WR1065 for a total of 35 min during irradiation with 60Co gamma-rays significantly reduces the incidence of neoplastic transformation while having no effect on cell viability. In a similar experiment with V79 cells in which 4 mM WR1065 was used, we found a significant reduction in mutation frequency at the HGPRT locus and significant protection against cell killing. These results suggest that WR1065 acts to modulate both acute damage and sub-lethal processes that lead to mutation and neoplastic transformation. Beyond the purely mechanistic approach of these studies, the potential application of these agents to minimizing the long-term neoplastic effects of radiation or chemotherapeutic agents currently in use for treating potentially curable cancer patients should be further investigated.
American Journal of Clinical Oncology, 1987
Sixty patients with high-grade gliomas (including 50 patients with glioblastoma multiforme) were ... more Sixty patients with high-grade gliomas (including 50 patients with glioblastoma multiforme) were entered on four sequential Phase I trials combining continuous intravenous infusions of halogenated pyrimidines and high-dose brain irradiation. Patients received two 14-day infusions of bromodeoxyuridine (BUdR) or iododeoxyuridine (IUdR) during the initial wide field and later reduced field radiation treatment (total radiation dose 65-70 Gy). All patients were
Cancer Research, 2018
Circadian clocks are intimately involved in the homeostatic maintenance of metabolic and physiolo... more Circadian clocks are intimately involved in the homeostatic maintenance of metabolic and physiological processes that may have increasingly important roles to enhance bone marrow transplantation success in cancer patients; to protect normal tissue surrounding tumors from the harmful effects of radio/chemotherapy; and to enhance the quality of diet and sleep for astronauts during space exploration. Herein, we report that expression of PERIOD2 (PER2; a core circadian clock component and “the inherent driver” of radioprotection) is required for adaptive protection against environmental radiation stress. PER2 expression is induced by exposure to low dose radiation (LDR; 10cGy) and siRNA blockade of PER2 ablates LDR-induced adaptive radioprotection. In addition, melatonin (N-acetyl-5-methoxytryptamine), a natural anti-oxidant and hypothalamic circadian synchronizer, stimulates PER2 expression and confers mice radioprotection through weight maintenance and increased survival. LDR-induced ...