Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin in which the variable heavy and light... more Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin in which the variable heavy and light domains of the anti-IL2 receptor antibody, anti-Tac, are connected to each other by a peptide linker and then fused to PE40, a truncated form of Pseudomonas exotoxin (PE). This fusion protein has four disulfide bonds: one in each of the two variables domains, one in domain II (Cys 265-287), and one in domain Ib (Cys 372-379) of PE. To study the importance of the disulfide bonds of the toxin to the activity of single-chain immunotoxins, we constructed mutants in which either the cysteines in the toxin were changed to alanines or the amino acids 365-380 of PE were deleted. We began this study with anti-Tac(Fv)-PE40 and a more active variant, anti-Tac(Fv)-PE40KDEL, in which the carbonyl terminus is changed from REDLK to KDEL. From these proteins we made anti-Tac(Fv)-PE40(4)A and anti-Tac(Fv)-PE40KDEL4A, respectively, by converting cysteins at amino acids 265, 287, 372, and 379 of PE to alanines. This change resulted in a 20-100-fold loss of activity toward human target cells, but no significant change in binding affinity to p55. To determine the importance of the second toxin disulfide bond, we removed amino acids 365-380 from anti-Tac(Fv)-PE40, anti-Tac(Fv)-PE40KDEL, and anti-Tac(Fv)-PE40KDEL4A, resulting in anti-Tac(Fv)-PE38, anti-Tac(Fv)-PE38KDEL, and anti-Tac(Fv)-PE38KDEL2A, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Patients with the variant form of xeroderma pigmentosum (XP) have clinical XP including a high fr... more Patients with the variant form of xeroderma pigmentosum (XP) have clinical XP including a high frequency of skin cancer but, in contrast to the other forms of XP, have normal post-ultraviolet (UV) DNA excision repair and nearly normal post-UV survival. However, like excision repair-deficient XP cells, the XP variant cells are UV hypermutable. We used a UV-treated plasmid shuttle vector,
Saraswathy Seetharam, Vijay K. ChaudharyS, David FitzGerald, and Ira Pastans From the Laboratory ... more Saraswathy Seetharam, Vijay K. ChaudharyS, David FitzGerald, and Ira Pastans From the Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health, Bethesda,-Maryland 20892
Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use o... more Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use of a shuttle vector plasmid, pZ189, containing a suppressor tRNA marker gene. In a series of experiments, 62 plasmids were recovered that had two to six base substitutions in the 160base-pair marker gene. Approximately 20-30% of the mutant plasmids that were recovered after passing ultraviolet-treated pZ189 through a repair-proficient human fibroblast line contained these multiple mutations. In contrast, passage of ultraviolet-treated pZ189 through an excision-repair-deficient (xeroderma pigmentosum) line yielded only 2% multiple base substitution mutants. Introducing a single-strand nick in otherwise unmodified pZ189 adjacent to the marker, followed by passage through the xeroderma pigmentosum cells, resulted in about 66% multiple base substitution mutants. The multiple mutations were found in a 160-base-pair region containing the marker gene but were rarely found in an adjacent 170-basepair region. Passing ultraviolet-treated or nicked pZ189 through a repair-proficient human B-cell line also yielded multiple base substitution mutations in 20-33% of the mutant plasmids. An explanation for these multiple mutations is that they were generated by an error-prone polymerase while filling gaps. These mutations share many of the properties displayed by mutations in the immunoglobulin hypervariable regions.
The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment... more The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment of collagen XVIII, enhances the antitumor effects of ionizing radiation (IR). Endostatin was injected to coincide with fractionated radiotherapy. Xenografts of radioresistant SQ-20B tumor cells were established in athymic nude mice. Lewis lung carcinoma cells were injected into C57BI/6 mice. Mice bearing SQ-20B xenografts were injected intraperitoneally with 2.5 mg/kg/day of murine recombinant endostatin 5 times per week for 2 weeks 3 hours before IR treatment (50 Gy total dose). Mice bearing Lewis lung carcinoma tumors were injected intraperitoneally with endostatin (2.5 mg/kg/day) four times; the first injection was given 24 hours before the first IR dose (15 Gy) and then 3 hours before IR (15 Gy/day) for 3 consecutive days. Microvascular density was assessed on tumor tissue sections by use of CD31 immunohistochemistry and light microscopy. Endothelial cell survival analyses were employed to evaluate endostatin effects on human aortic endothelial cells and human umbilical vein endothelial cells. Endothelial cell apoptosis was examined by use of FACS analysis and DAPI microscopy. In SQ-20B xenografts, combined treatment with endostatin and IR produced tumor growth inhibition that was most pronounced at the nadir of regression (day 21). By day 35, tumors receiving combined treatment with endostatin and IR were 47% smaller than tumors treated with endostatin alone. Interactive cytotoxic treatment effects between endostatin and IR were also demonstrated in mice bearing Lewis lung carcinoma tumors. Significant tumor growth inhibition was observed in the endostatin/IR group at days 11 and 13 compared with IR alone. Histologic analyses demonstrated a reduction in microvascular density after combined treatment with endostatin and IR compared with endostatin treatment alone. Survival analyses confirmed interactive cytotoxicity between endostatin and IR in both human aortic endothelial cells and human umbilical vein endothelial cells but not in SQ-20B tumor cells. Combined treatment with endostatin and IR produced an increase in cow pulmonary artery endothelial apoptosis compared with either treatment alone. The tumor regression observed after combined treatment with endostatin and IR suggests additive antitumor effects in both human and murine tumors. Importantly, the concentrations of endostatin employed produced little tumor regression when endostatin was employed as a single agent. The results from the clonogenic and apoptosis assays support the hypothesis that the endothelial compartment is the target for the endostatin/IR interaction.
Metabolic effects of cortisol may be critically modulated by glucocorticoid metabolism in tissues... more Metabolic effects of cortisol may be critically modulated by glucocorticoid metabolism in tissues. Specifically, active cortisol is regenerated from inactive cortisone by the enzyme 11-hydroxysteroid dehydrogenase type 1 (11-HSD1) in adipose and liver. We examined activity and mRNA levels of 11-HSD1 and tissue cortisol and cortisone levels in sc adipose tissue biopsies from 12 Caucasian (7 males and 5 females) and 19 Pima Indian (10 males and 9 females) nondiabetic subjects aged 28 ؎ 7.6 yr (mean ؎ SD; range, 18 -45). Adipose 11-HSD1 activity and mRNA levels were highly correlated (r ؍ 0.51, P ؍ 0.003). Adipose 11-HSD1 activity was positively related to measures of total (body mass index, percentage body fat) and cen-tral (waist circumference) adiposity (P < 0.05 for all) and fasting glucose (r ؍ 0.43, P ؍ 0.02), insulin (r ؍ 0.60, P ؍ 0.0005), and insulin resistance by the homeostasis model (r ؍ 0.70, P < 0.0001) but did not differ between sexes or ethnic groups. Intra-adipose cortisol was positively associated with fasting insulin (r ؍ 0.37, P ؍ 0.04) but was not significantly correlated with 11-HSD1 mRNA or activity or with other metabolic variables. In this cross-sectional study, higher adipose 11-HSD1 activity is associated with features of the metabolic syndrome. Our data support the hypothesis that increased regeneration of cortisol in adipose tissue influences metabolic sequelae of human obesity. (J Clin Endocrinol
The role of UV radiation-induced photoproducts in initiating base substitution mutations in human... more The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. AU transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of >99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosinecontaining non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.
The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mito... more The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mitogens for vascular endothelial cells that function in the regulation of angiogenesis. Inhibition of VEGF-induced angiogenesis, either by neutralizing antibodies or a dominant-negative soluble receptor, blocks the growth of primary and metastatic experimental tumors. Here we report that VEGF expression is induced in Lewis lung carcinomas (LLCs) both in vitro and in vivo after exposure to ionizing radiation (IR) and in human tumor cell lines (Seg-1 esophageal adenocarcinoma, SQ20B squamous cell carcinoma, T98 and U87 glioblastomas, and U1 melanoma) in vitro. The biological significance of IR-induced VEGF production is supported by our finding that treatment of tumor-bearing mice (LLC, Seg-1, SQ20B, and U87) with a neutralizing antibody to VEGF-165 before irradiation is associated with a greater than additive antitumor effect.
Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin in which the variable heavy and light... more Anti-Tac(Fv)-PE40 is a recombinant single-chain immunotoxin in which the variable heavy and light domains of the anti-IL2 receptor antibody, anti-Tac, are connected to each other by a peptide linker and then fused to PE40, a truncated form of Pseudomonas exotoxin (PE). This fusion protein has four disulfide bonds: one in each of the two variables domains, one in domain II (Cys 265-287), and one in domain Ib (Cys 372-379) of PE. To study the importance of the disulfide bonds of the toxin to the activity of single-chain immunotoxins, we constructed mutants in which either the cysteines in the toxin were changed to alanines or the amino acids 365-380 of PE were deleted. We began this study with anti-Tac(Fv)-PE40 and a more active variant, anti-Tac(Fv)-PE40KDEL, in which the carbonyl terminus is changed from REDLK to KDEL. From these proteins we made anti-Tac(Fv)-PE40(4)A and anti-Tac(Fv)-PE40KDEL4A, respectively, by converting cysteins at amino acids 265, 287, 372, and 379 of PE to alanines. This change resulted in a 20-100-fold loss of activity toward human target cells, but no significant change in binding affinity to p55. To determine the importance of the second toxin disulfide bond, we removed amino acids 365-380 from anti-Tac(Fv)-PE40, anti-Tac(Fv)-PE40KDEL, and anti-Tac(Fv)-PE40KDEL4A, resulting in anti-Tac(Fv)-PE38, anti-Tac(Fv)-PE38KDEL, and anti-Tac(Fv)-PE38KDEL2A, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Patients with the variant form of xeroderma pigmentosum (XP) have clinical XP including a high fr... more Patients with the variant form of xeroderma pigmentosum (XP) have clinical XP including a high frequency of skin cancer but, in contrast to the other forms of XP, have normal post-ultraviolet (UV) DNA excision repair and nearly normal post-UV survival. However, like excision repair-deficient XP cells, the XP variant cells are UV hypermutable. We used a UV-treated plasmid shuttle vector,
Saraswathy Seetharam, Vijay K. ChaudharyS, David FitzGerald, and Ira Pastans From the Laboratory ... more Saraswathy Seetharam, Vijay K. ChaudharyS, David FitzGerald, and Ira Pastans From the Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health, Bethesda,-Maryland 20892
Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use o... more Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use of a shuttle vector plasmid, pZ189, containing a suppressor tRNA marker gene. In a series of experiments, 62 plasmids were recovered that had two to six base substitutions in the 160base-pair marker gene. Approximately 20-30% of the mutant plasmids that were recovered after passing ultraviolet-treated pZ189 through a repair-proficient human fibroblast line contained these multiple mutations. In contrast, passage of ultraviolet-treated pZ189 through an excision-repair-deficient (xeroderma pigmentosum) line yielded only 2% multiple base substitution mutants. Introducing a single-strand nick in otherwise unmodified pZ189 adjacent to the marker, followed by passage through the xeroderma pigmentosum cells, resulted in about 66% multiple base substitution mutants. The multiple mutations were found in a 160-base-pair region containing the marker gene but were rarely found in an adjacent 170-basepair region. Passing ultraviolet-treated or nicked pZ189 through a repair-proficient human B-cell line also yielded multiple base substitution mutations in 20-33% of the mutant plasmids. An explanation for these multiple mutations is that they were generated by an error-prone polymerase while filling gaps. These mutations share many of the properties displayed by mutations in the immunoglobulin hypervariable regions.
The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment... more The purpose of this study was to evaluate whether endostatin, an antiangiogenic cleavage fragment of collagen XVIII, enhances the antitumor effects of ionizing radiation (IR). Endostatin was injected to coincide with fractionated radiotherapy. Xenografts of radioresistant SQ-20B tumor cells were established in athymic nude mice. Lewis lung carcinoma cells were injected into C57BI/6 mice. Mice bearing SQ-20B xenografts were injected intraperitoneally with 2.5 mg/kg/day of murine recombinant endostatin 5 times per week for 2 weeks 3 hours before IR treatment (50 Gy total dose). Mice bearing Lewis lung carcinoma tumors were injected intraperitoneally with endostatin (2.5 mg/kg/day) four times; the first injection was given 24 hours before the first IR dose (15 Gy) and then 3 hours before IR (15 Gy/day) for 3 consecutive days. Microvascular density was assessed on tumor tissue sections by use of CD31 immunohistochemistry and light microscopy. Endothelial cell survival analyses were employed to evaluate endostatin effects on human aortic endothelial cells and human umbilical vein endothelial cells. Endothelial cell apoptosis was examined by use of FACS analysis and DAPI microscopy. In SQ-20B xenografts, combined treatment with endostatin and IR produced tumor growth inhibition that was most pronounced at the nadir of regression (day 21). By day 35, tumors receiving combined treatment with endostatin and IR were 47% smaller than tumors treated with endostatin alone. Interactive cytotoxic treatment effects between endostatin and IR were also demonstrated in mice bearing Lewis lung carcinoma tumors. Significant tumor growth inhibition was observed in the endostatin/IR group at days 11 and 13 compared with IR alone. Histologic analyses demonstrated a reduction in microvascular density after combined treatment with endostatin and IR compared with endostatin treatment alone. Survival analyses confirmed interactive cytotoxicity between endostatin and IR in both human aortic endothelial cells and human umbilical vein endothelial cells but not in SQ-20B tumor cells. Combined treatment with endostatin and IR produced an increase in cow pulmonary artery endothelial apoptosis compared with either treatment alone. The tumor regression observed after combined treatment with endostatin and IR suggests additive antitumor effects in both human and murine tumors. Importantly, the concentrations of endostatin employed produced little tumor regression when endostatin was employed as a single agent. The results from the clonogenic and apoptosis assays support the hypothesis that the endothelial compartment is the target for the endostatin/IR interaction.
Metabolic effects of cortisol may be critically modulated by glucocorticoid metabolism in tissues... more Metabolic effects of cortisol may be critically modulated by glucocorticoid metabolism in tissues. Specifically, active cortisol is regenerated from inactive cortisone by the enzyme 11-hydroxysteroid dehydrogenase type 1 (11-HSD1) in adipose and liver. We examined activity and mRNA levels of 11-HSD1 and tissue cortisol and cortisone levels in sc adipose tissue biopsies from 12 Caucasian (7 males and 5 females) and 19 Pima Indian (10 males and 9 females) nondiabetic subjects aged 28 ؎ 7.6 yr (mean ؎ SD; range, 18 -45). Adipose 11-HSD1 activity and mRNA levels were highly correlated (r ؍ 0.51, P ؍ 0.003). Adipose 11-HSD1 activity was positively related to measures of total (body mass index, percentage body fat) and cen-tral (waist circumference) adiposity (P < 0.05 for all) and fasting glucose (r ؍ 0.43, P ؍ 0.02), insulin (r ؍ 0.60, P ؍ 0.0005), and insulin resistance by the homeostasis model (r ؍ 0.70, P < 0.0001) but did not differ between sexes or ethnic groups. Intra-adipose cortisol was positively associated with fasting insulin (r ؍ 0.37, P ؍ 0.04) but was not significantly correlated with 11-HSD1 mRNA or activity or with other metabolic variables. In this cross-sectional study, higher adipose 11-HSD1 activity is associated with features of the metabolic syndrome. Our data support the hypothesis that increased regeneration of cortisol in adipose tissue influences metabolic sequelae of human obesity. (J Clin Endocrinol
The role of UV radiation-induced photoproducts in initiating base substitution mutations in human... more The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. AU transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of >99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosinecontaining non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.
The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mito... more The family of vascular endothelial growth factor (VEGF) proteins include potent and specific mitogens for vascular endothelial cells that function in the regulation of angiogenesis. Inhibition of VEGF-induced angiogenesis, either by neutralizing antibodies or a dominant-negative soluble receptor, blocks the growth of primary and metastatic experimental tumors. Here we report that VEGF expression is induced in Lewis lung carcinomas (LLCs) both in vitro and in vivo after exposure to ionizing radiation (IR) and in human tumor cell lines (Seg-1 esophageal adenocarcinoma, SQ20B squamous cell carcinoma, T98 and U87 glioblastomas, and U1 melanoma) in vitro. The biological significance of IR-induced VEGF production is supported by our finding that treatment of tumor-bearing mice (LLC, Seg-1, SQ20B, and U87) with a neutralizing antibody to VEGF-165 before irradiation is associated with a greater than additive antitumor effect.
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