End-labeled DNA Fragments Research Articles

Oxidative DNA damage induced by photodegradation products of 3 ′-azido-3 ′-deoxythymidine

3 ′-Azido-3 ′-deoxythymidine (AZT) is carcinogenic to experimental animals and can cause the formation of 8-oxo-7,8-dihydro-2 ′-deoxyguanosine (8-oxodG) in humans and animals. To clarify the mechanism of carcinogenesis by AZT, we investigated DNA damage induced by its photodegradation products, using 32P-5 ′-end-labeled DNA fragments obtained from human genes. Following exposure to UVB, AZT induced DNA damage in the presence of Cu(II). Catalase inhibited DNA damage, indicating the involvement of H 2O 2. UVB-exposed AZT plus Cu(II) induced 8-oxodG formation in a dose-dependent manner. Mass spectrum of UVB-exposed AZT demonstrated the generation of a hydroxylamine derivative. The colorimetric determination suggested that AZT was converted into the hydroxylamine derivative depending on UVB doses. UVB-exposed AZT induced double base damage at the 5 ′-A CG -3 ′ sequence, complementary to a hot spot of the p53 gene. The basic compound, hydroxylamine, showed similar site specificity. The hydroxylamine derivative produced by photodegradation and/or possible metabolism of AZT induces oxidative DNA damage, which may participate in carcinogenesis.

Recruitment of σ54-RNA Polymerase to the Pu Promoter of Pseudomonas putida through Integration Host Factor-mediated Positioning Switch of α Subunit Carboxyl-terminal Domain on an UP-like Element

The interactions between the sigma54-containing RNA polymerase (sigma54-RNAP) and the region of the Pseudomonas putida Pu promoter spanning from the enhancer to the binding site for the integration host factor (IHF) were analyzed both by DNase I and hydroxyl radical footprinting. A short Pu region centered at position -104 was found to be involved in the interaction with sigma54-RNAP, both in the absence and in the presence of IHF protein. Deletion or scrambling of the -104 region strongly reduced promoter affinity in vitro and promoter activity in vivo, respectively. The reduction in promoter affinity coincided with the loss of IHF-mediated recruitment of the sigma54-RNAP in vitro. The experiments with oriented-alpha sigma54-RNAP derivatives containing bound chemical nuclease revealed interchangeable positioning of only one of the two alpha subunit carboxyl-terminal domains (alphaCTDs) both at the -104 region and in the surroundings of position -78. The addition of IHF resulted in perfect position symmetry of the two alphaCTDs. These results indicate that, in the absence of IHF, the sigma54-RNAP asymmetrically uses only one alphaCTD subunit to establish productive contacts with upstream sequences of the Pu promoter. In the presence of IHF-induced curvature, the closer proximity of the upstream DNA to the body of the sigma54-RNAP can allow the other alphaCTD to be engaged in and thus favor closed complex formation.

Effects of caspase inhibition on the progression of experimental glomerulonephritis

Caspase-3 has a central role in the execution of apoptosis. In a nephrotoxic nephritis (NTN) model, we previously demonstrated an up-regulation of caspase-3 that was associated with inappropriate renal apoptosis, inflammation, tubular atrophy, and renal scarring. We applied a pan caspase inhibitor, Boc-Asp (OMe)-fluoro-methyl-ketone (B-D-FMK), directly to rat NTN kidney using an intrarenal cannula fed from an osmotic pump. Animals were treated either for the first 7 days (acutely) to determine the effects on renal inflammation (ED-1 staining) and apoptosis (in situ end labeling of fragmented DNA), or for 28 days commencing 15 days after NTN (chronically) to observe the effects on cell death and renal fibrosis. Changes of caspase-3 and caspase-1 activity were detected by fluorometric substrate cleavage assay. Changes in caspase-3 and caspase-1, interleukin-1 beta (IL-1 beta), and collagen I, III, and IV proteins and mRNA were detected by Western blotting and Northern blotting, respectively. In both treated groups, caspase-3 activity was inhibited, and 17 and 24 kD active caspase-3 proteins were reduced significantly. A compensatory increase of caspase-3 mRNA occurred in the acutely treated group, but decreased in the chronically treated group (P < 0.05). Although there were no significant changes in caspase-1 activity and its active protein, the observed decrease in its precursor in the chronic group was increased by treatment (P < 0.05). Further, IL-1 beta precursor and its mRNA were significantly reduced by treatment only in the chronically treated group. Apoptosis was decreased in the glomeruli of acutely treated rats, and in the tubules and interstitium of chronically treated animals (P < 0.05). Glomerular inflammation was decreased only in the acutely treated group, whereas tubulointerstitial inflammation was lowered in both treated groups (P < 0.05). Glomerulosclerosis was reduced in both inhibitor groups, with a reduction in tubulointerstitial fibrosis and collagen I, III, and IV mRNA restricted to chronically treated animals (P < 0.05). Proteinuria was significantly decreased with caspase inhibition in both treated groups, but not serum creatinine level. This study clearly indicates that caspase inhibition reduces renal apoptosis, ameliorates inflammation and fibrosis, and improves proteinuria in experimental glomerulonephritis, which may mainly be related to changes in the caspase enzymatic system.

Changes in the topological expression of markers of differentiation and apoptosis in defined stages of human cervical dysplasia and carcinoma

Objective We compared the capacity of cells in normal cervical epithelium, progressive stages of CIN, and invasive carcinoma to proliferate, differentiate, and undergo apoptosis. Methods We investigated 30 conizations showing regular squamous epithelium of the ectocervix, all stages of cervical preinvasive neoplastic lesions (CIN I to III), or invasive carcinoma. The expression of the cell proliferation and differentiation marker Ki67 and Mad-1, respectively, and of the apoptosis-related proteins bcl-2, active caspase-3, and DNase I was analyzed on paraffin sections by immunohistochemistry. The expression of DNase I or -like enzymes was also analyzed at the level of their gene transcripts by in situ hybridization. In addition, apoptotic events were identified by in situ end labeling of fragmented DNA (ISEL). Results Expression of Ki67 was restricted to suprabasal cells in normal cervical epithelium but increased with CIN severity and invasive carcinoma. ISEL demonstrated apoptosis in superficial layers of normal, CIN I, and CIN II epithelium, whereas in CIS (CIN III) and invasive carcinoma, ISEL-positive cells were additionally observed at varying epithelial locations. Bcl-2 immunostaining remained restricted to the basal layer of all preneoplastic and neoplastic stages. Active caspase-3 was present in the suprabasal layer and extended to all upper layers in normal epithelium and slightly decreased with increasing dysplasia. In invasive carcinoma it was restricted to few scattered cells. The differentiation marker Mad-1 extended from the spinous to the superficial layer in regular epithelium, but gradually shifted to more superficial layers with increasing CIN grade and invasive carcinoma. A similar topological change was observed for DNase I with increasing CIN grade. In CIS and invasive carcinoma, DNase I immunopositive cells were solely interspersed within neoplastic cells. In contrast, DNase I specific mRNA was present in all epithelial layers in CIN III and neoplasia, suggesting a translational block of the expression of DNase I or -like enzymes. Conclusions Our data indicate that the elevated proliferation observed with increasing CIN severity and carcinoma was not paralleled by a similar increase in cell elimination. Most of the dysplastic and neoplastic cervical epithelial cells appeared incapable of entering terminal differentiation and complete it by apoptosis, possibly due to their failure to express or activate apoptosis executing enzymes.

A phenylacetate derivative, SCK6, inhibits cell proliferation via G 1 cell cycle arrest and apoptosis

Phenylacetate is a differentiation agent and has anticancer activity with relatively low toxicity. In the present study, we examined the anticancer effect of six synthetic phenylacetate derivatives in human lung cancer cells in our search for more effective phenylacetate analogous. Results showed that the antiproliferative effects of these synthetic compounds were stronger than those of phenylacetate, and that N-butyl-2-(2-fluorolphenyl)acetamide (SCK6) is the most potent compound. To address the mechanism of the antiproliferative effect of SCK6, cell cycle analysis was performed. Result showed that SCK6 (1 mM) induced G 1 arrest in CH27 cells. Western blot analysis of G 1 phase regulatory proteins demonstrated that the protein levels of cyclin-dependent kinase 2 (Cdk2), Cdk4, Cyclin E and Cyclin D3 were decreased after treatment with SCK6 but not those of Cdk6, Cyclin D1 and D2. In contrast, SCK6 increased the protein levels of p53 and p21 CIP1/WAF1. Data from in situ terminal transferase-mediated dUTP-fluorescensin nick end-labeling (TUNEL) assay and DNA fragmentation analysis demonstrated that SCK6 induced apoptotic cell death in CH27 cells. This SCK6-induced apoptosis was accompanied by a downregulation of Bcl-2 protein and activation of the caspase-9 cascade. Overexpression of Bcl-2 by adeno-Bcl-2 vector infection significantly inhibited SCK6-induced apoptosis. Moreover, treatment with caspase inhibitors also markedly reduced cell death induced by SCK6. Taken together, these results suggest that downregulation of G 1-associated Cdks and cyclins and upregulation of p53 and p21 CIP1/WAF1 may contribute to SCK6-mediated G 1-phase arrest. Furthermore, the decrease in Bcl-2 and the activation of caspase-9/caspase-3 may be the effector mechanism through which SCK6 induces apoptosis.

Carcinogenic semicarbazide induces sequence-specific DNA damage through the generation of reactive oxygen species and the derived organic radicals

Semicarbazide, a hydrazine derivative, is carcinogenic to mice but shows no or little mutagenicity in the Salmonella-microsome test. To clarify whether or not the genotoxic mechanism contributes to the non-mutagenic carcinogenicity of semicarbazide, we investigated DNA damage induced by semicarbazide using 32 P -5′-end-labeled DNA fragments obtained from the c-Ha- ras-1 protooncogene and the p53 tumor suppressor gene. Semicarbazide caused DNA damage frequently at the thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine partially inhibited DNA damage, suggesting that hydrogen peroxide plus Cu(I) participates in DNA damage. When a high concentration of semicarbazide was used in the presence of catalase, DNA damage was induced, especially at G in 5′-AG and slightly at 5′-G in GG and GGG sequences. An electron paramagnetic resonance (EPR) spectroscopic study has confirmed that the reaction of semicarbazide with Cu(II) produces carbamoyl radicals ( CONH 2), possibly generated via the nitrogen-centered radicals of semicarbazide. Azodicarbonamide also produced carbamoyl radicals and induced DNA damage frequently at 5′-G in GG and GGG sequences, suggesting that carbamoyl radicals participate in this sequence-specific DNA damage by semicarbazide. On the basis of our previous reports, we consider that the sequence-specific DNA damage at G in 5′-AG in the present study is due to the nitrogen-centered radicals. This study has shown that semicarbazide induces DNA damage in the presence of Cu(II) through the formation of hydrogen peroxide and Cu(I). In addition, semicarbazide-derived free radicals participate in DNA damage. DNA damage induced by these reactive species may be relevant to the carcinogenicity of semicarbazide.

Apoptosis and Regression of Embryonic Mesenchyme in the Development of the Middle Ear Spaces

Apoptosis was studied using temporal bones from three fetuses representing different times of gestation and from three neonates. Paraffin-embedded sections 20-μm thick were studied using the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling method based on 3′-end-labeling of fragmented DNA. Phenotyping of the immune cells was performed using regular monoclonal antibodies. In the bone marrow the granulocyte series dominated and the number of cells in the macrophage series was noticeably fewer, with apoptotic cells occurring in both. In the embryonic mesenchyme, solitary apoptotic cells occurred in all locations in both the fetuses and neonates. Apoptosis is a basic factor in the regression of embryonal mesenchyme, but may not be preprogrammed. Basic scientific data obtained from modified tissue cultures show that mechanical forces cause cells to switch between different genetic programs. It is suggested that the act of swallowing causes periodic changes in the amniotic fluid pressure and provides the necessary force for regression of the mesenchyme by apoptosis.

Expression of Bcl-2 and Bax in rhesus monkey testis during germ cell apoptosis induced by testosterone undecanoate.

Apoptosis occurs spontaneously during spermatogenesis and can be induced by androgen withdrawal. However, the molecular events governing apoptosis have not been characterized. To study the molecular mechanism of apoptosis induced by a high dose of testosterone undecanoate (TU), the authors examined the temporal changes in proapoptotic Bax and antiapoptotic Bcl-2 in TU-treated monkey testes. Apoptotic cells were identified in tissue sections by in situ end labeling of fragmented DNA. The results showed that a great deal of the apoptotic cells occurred in the testes on day 30 after TU injection and that the dominant apoptotic germ cells are spermatocytes and spermatids. The expression of Bcl-2 and Bax was assessed by immunohistochemical method and Western blot. As compared with that of normal testes, the levels of Bcl-2 protein increased significantly from 7 to day 14 while that of Bax protein was almost unchanged in the testes from day 7 up to day 60 after TU treatment. Bcl-2 was localized to the spermatids in the normal testes and temporarily distributed in both the cytoplasm and nucleus of those cell types susceptible to TU-induced apoptosis on day 14 after TU injection. Therefore, it is suggested that Bax may not play a role in initiating germ cell apoptosis induced by TU injection and that the evaluation in Bcl-2 expression may represent a survival mechanism for the remaining germ cell.

Apoptosis and microglial activation in influenza encephalopathy.

During influenza epidemics in Japan, the number of children with acute encephalopathies and encephalitis has recently increased. Although the pathophysiologies remain unclear, there is usually brain edema with evidence of damage to the blood-brain-barrier (BBB). We investigated the glial reaction and apoptosis in brains of eight such cases comprising two of acute necrotizing encephalopathy and six of influenza encephalopathy, and compared the results with those in five control brains. Apoptosis, evidenced by chromatin condensation and fragmentation in hematoxylin sections, in situ end labeling of fragmented DNA (TUNEL) and DNA laddering, was observed in neurons and glial cells in four brains with influenza encephalopathy. In the TUNEL-positive brains, the increase in microglia was greater than in the TUNEL-negative brains. Immunoreactivity for active-caspase 3, demonstrated by immunohistochemistry, and the overexpression of a caspase-cleaved fragment of poly(ADP-ribose) polymerase, demonstrated by Western blotting, indicated that activation of caspase 3 is involved in the apoptotic pathway in the brains of influenza encephalopathy cases. Apoptosis or specific pathological processes that cause apoptosis may give rise to aggravated encephalopathy.

Aryl hydrocarbon receptor regulates growth, but not atresia, of mouse preantral and antral follicles.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that binds various environmental contaminants. Despite our knowledge regarding the role of the AhR in mediating toxicity, little is known about the physiological role of the AhR. Previous studies indicate that the AhR may regulate folliculogenesis, because AhR-deficient (AhRKO) mice have fewer preantral and antral follicles than wild-type (WT) mice during postnatal life. Thus, the first objective of the present study was to test the hypothesis that AhR deficiency reduces the numbers of preantral and antral follicles by slowing growth and/or increasing atresia of follicles. Because alterations in follicular growth or atresia can affect the ability to ovulate, the second objective was to test whether AhR deficiency reduces the number of ovulated eggs. To test these hypotheses, follicular growth was compared in WT and AhRKO ovaries using morphometric techniques and by measuring the ability of the ovary and follicles to grow in response to eCG. Atresia was compared in WT and AhRKO ovaries using morphometric techniques, TUNEL assays, and 3'-end labeling of fragmented DNA. Ovulation was compared in WT and AhRKO mice by assessing the number of corpora lutea per ovary. The results indicate that follicular growth and ovulation were reduced in AhRKO ovaries compared to WT ovaries. The WT ovaries had a 1.5-fold increase in the number of preantral and antral follicles between Postnatal Days 32 and 45, were more responsive to eCG, and contained more corpora lutea than AhRKO ovaries. In contrast, no significant difference was observed in the incidence of atresia in WT and AhRKO ovaries. Taken together, these results suggest that the AhR may regulate growth, but not atresia, of preantral and antral follicles in the mouse ovary.

A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis

A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis.BackgroundAlthough apoptosis has been linked to the renal cell deletion and ensuing renal fibrosis, its regulating mechanisms remain obscure. Of the known regulators of apoptosis, the best characterized is the Bax to Bcl-2 ratio. However, its importance in controlling apoptosis in glomerulonephritis is unclear. Here, using the nephrotoxic nephritis (NTN) model, we evaluated Bax/Bcl-2 in relation to changes in the apoptosis co-ordination enzyme, caspase-3.MethodsKidneys were harvested at days 7, 15, 30 and 45 post-injection of anti-glomerular basement membrane antibody into Wistar Kyoto rats. These were analyzed for apoptosis (in situ end labeling of fragmented DNA, light and electron microscopy), Bax/Bcl-2 protein (Western blotting), mRNA (Northern blotting) and distribution (immunohistochemistry), as well as caspase-3 activity (substrate cleavage assay), inflammation (ED1 staining), proliferation (proliferating cell nuclear antigen staining) and fibrosis (Masson's Trichrome staining).ResultsBax mRNA was significantly increased while that of Bcl-2 was decreased throughout the time course (+265% and -62% by day 45). Increased Bax and decreased Bcl-2 protein were noted, significantly so on day 7 (+177% and -21%) and day 45 (+363% and -17%). Bax protein was observed in dilated and atrophic tubules, sclerotic glomeruli and inflamed interstitium, while Bcl-2 was only visible in atrophic tubules. The ratios of Bax to Bcl-2 mRNA and protein were significantly increased at all time points. These correlated (P < 0.05) with up-regulated caspase-3 activity (r = 0.742 and 0.531), apoptosis (r = 0.712 and 0.540), proliferation (r = 0.611, mRNA only), inflammation (ED1+, r = 0.474 and 0.419) and fibrosis (r = 0.474 and 0.729).ConclusionsOur findings suggest that the changes in the ratio of Bax to Bcl-2 may contribute to the caspase-3 activation and the modulation of renal apoptosis associated with renal inflammation, tubular atrophy and renal fibrosis in experimental glomerulonephritis. A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis. Although apoptosis has been linked to the renal cell deletion and ensuing renal fibrosis, its regulating mechanisms remain obscure. Of the known regulators of apoptosis, the best characterized is the Bax to Bcl-2 ratio. However, its importance in controlling apoptosis in glomerulonephritis is unclear. Here, using the nephrotoxic nephritis (NTN) model, we evaluated Bax/Bcl-2 in relation to changes in the apoptosis co-ordination enzyme, caspase-3. Kidneys were harvested at days 7, 15, 30 and 45 post-injection of anti-glomerular basement membrane antibody into Wistar Kyoto rats. These were analyzed for apoptosis (in situ end labeling of fragmented DNA, light and electron microscopy), Bax/Bcl-2 protein (Western blotting), mRNA (Northern blotting) and distribution (immunohistochemistry), as well as caspase-3 activity (substrate cleavage assay), inflammation (ED1 staining), proliferation (proliferating cell nuclear antigen staining) and fibrosis (Masson's Trichrome staining). Bax mRNA was significantly increased while that of Bcl-2 was decreased throughout the time course (+265% and -62% by day 45). Increased Bax and decreased Bcl-2 protein were noted, significantly so on day 7 (+177% and -21%) and day 45 (+363% and -17%). Bax protein was observed in dilated and atrophic tubules, sclerotic glomeruli and inflamed interstitium, while Bcl-2 was only visible in atrophic tubules. The ratios of Bax to Bcl-2 mRNA and protein were significantly increased at all time points. These correlated (P < 0.05) with up-regulated caspase-3 activity (r = 0.742 and 0.531), apoptosis (r = 0.712 and 0.540), proliferation (r = 0.611, mRNA only), inflammation (ED1+, r = 0.474 and 0.419) and fibrosis (r = 0.474 and 0.729). Our findings suggest that the changes in the ratio of Bax to Bcl-2 may contribute to the caspase-3 activation and the modulation of renal apoptosis associated with renal inflammation, tubular atrophy and renal fibrosis in experimental glomerulonephritis.

A shift in the Bax/Bcl-2 balance may activate caspase-3 and modulate apoptosis in experimental glomerulonephritis

Although apoptosis has been linked to the renal cell deletion and ensuing renal fibrosis, its regulating mechanisms remain obscure. Of the known regulators of apoptosis, the best characterized is the Bax to Bcl-2 ratio. However, its importance in controlling apoptosis in glomerulonephritis is unclear. Here, using the nephrotoxic nephritis (NTN) model, we evaluated Bax/Bcl-2 in relation to changes in the apoptosis co-ordination enzyme, caspase-3. Kidneys were harvested at days 7, 15, 30 and 45 post-injection of anti-glomerular basement membrane antibody into Wistar Kyoto rats. These were analyzed for apoptosis (in situ end labeling of fragmented DNA, light and electron microscopy), Bax/Bcl-2 protein (Western blotting), mRNA (Northern blotting) and distribution (immunohistochemistry), as well as caspase-3 activity (substrate cleavage assay), inflammation (ED1 staining), proliferation (proliferating cell nuclear antigen staining) and fibrosis (Masson's Trichrome staining). Bax mRNA was significantly increased while that of Bcl-2 was decreased throughout the time course (+265% and -62% by day 45). Increased Bax and decreased Bcl-2 protein were noted, significantly so on day 7 (+177% and -21%) and day 45 (+363% and -17%). Bax protein was observed in dilated and atrophic tubules, sclerotic glomeruli and inflamed interstitium, while Bcl-2 was only visible in atrophic tubules. The ratios of Bax to Bcl-2 mRNA and protein were significantly increased at all time points. These correlated (P < 0.05) with up-regulated caspase-3 activity (r = 0.742 and 0.531), apoptosis (r = 0.712 and 0.540), proliferation (r = 0.611, mRNA only), inflammation (ED1+, r = 0.474 and 0.419) and fibrosis (r = 0.474 and 0.729). Our findings suggest that the changes in the ratio of Bax to Bcl-2 may contribute to the caspase-3 activation and the modulation of renal apoptosis associated with renal inflammation, tubular atrophy and renal fibrosis in experimental glomerulonephritis.

Apoptosis and Myofibroblast Expression in Human Glomerular Disease: A Possible Link with Transforming Growth Factor-Beta-1

Background/Aims: The pathophysiological pathways involved in the pathogenesis and evolution of renal fibrosis, have not been fully elucidated. Transforming growth factor-beta₁ (TGF-β₁) is involved in the development of renal scarring. Apoptosis is responsible for intrinsic cell deletion observed in end-stage kidney disease. Myofibroblasts are involved in the development of renal fibrosis. This study investigates whether there is a potential relationship between apoptosis, myofibroblast infiltration and TGF-β₁ expression in the kidney of patients with glomerulonephritis (GN). Methods: Forty patients with various types of GN were included in the study. Myofibroblasts and TGF-β₁ positive cells were detected in kidney biopsies by immunohistochemistry, while apoptotic cells were detected by the in situ end labelling of fragmented DNA. Results: Myofibroblasts were identified in the glomeruli of some patients with severe mesangioproliferative GN and glomerulosclerosis but a more intensive myofibroblast expression was found in the renal interstitium. TGF-β₁ was expressed in the cytoplasm of tubular epithelial cells, in the renal interstitium and in the glomeruli of patients with GN. Apoptotic cells were mainly detected in the tubules and interstitium and were present in areas with intense myofibroblast infiltration. Positive correlations were observed between the intensity of myofibroblast expression in the interstitium and apoptosis in the tubulointerstitial area (r = 0.521, p < 0.01) as well as TGF-β₁ expression (r = 0.462, p < 0.05) and degree of renal impairment (r = 0.430, p < 0.05). Conclusions: These observations suggest that myofibroblast infiltration and apoptosis along with TGF-β₁ expression are associated with the development of interstitial fibrosis in patients with glomerular disease.

Metal-mediated DNA damage induced by curcumin in the presence of human cytochrome P450 isozymes

Although curcumin is known to exhibit antitumor activity, carcinogenic properties have also been reported. To clarify the potentiality of carcinogenesis by curcumin, we have examined whether curcumin can induce DNA damage in the presence of cytochrome P450 (CYP) using [ 32 P] -5 ′ -end-labeled DNA fragments obtained from genes relevant to human cancer. Curcumin treated with CYP 2D6, CYP1A1, or CYP1A2 induced DNA damage in the presence of Cu(II). CYP2D6-treated curcumin caused base damage, especially at 5 ′-TG-3 ′, 5 ′-GC-3 ′, and GG sequences. The DNA damage was inhibited by both catalase and bathocuproine, suggesting that reactive species derived from the reaction of H 2O 2 with Cu(I) participate in DNA damage. Formation of 8-oxo-7,8-dihydro-2 ′-deoxyguanosine was significantly increased by CYP2D6-treated curcumin in the presence of Cu(II). Time-of- flight mass spectrometry demonstrated that CYP2D6 catalyzed the conversion of curcumin to O-demethyl curcumin. Therefore, it is concluded that curcumin may exhibit carcinogenic potential through oxidative DNA damage by its metabolite.

Metabolism of carcinogenic urethane to nitric oxide is involved in oxidative DNA damage

Carcinogenic urethane (ethyl carbamate) forms DNA adduct via epoxide, whereas carcinogenic methyl carbamate can not. To clarify a mechanism independent of DNA adduct formation, we examined DNA damage induced by N-hydroxyurethane, a urethane metabolite, using 32P-5′-end-labeled DNA fragments. N-hydroxyurethane induced Cu(II)-mediated DNA damage especially at thymine and cytosine residues. DNA damage was inhibited by both catalase and bathocuproine, suggesting a role for H 2O 2 and Cu(I) in DNA damage. Free OH scavengers did not inhibit the DNA damage, although methional did inhibit it. These results suggest that reactive species, such as the Cu(I)-hydroperoxo complex, cause DNA damage. Formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) was increased by N-hydroxyurethane in the presence of Cu(II). When treated with esterase, N-hydroxyurethane induced 8-oxodG formation to a similar extent as that induced by hydroxylamine. Enhancement of DNA cleavages by endonuclease IV suggests that hydroxylamine induced depurination. Furthermore, hydroxylamine induced a significant increase in 8-oxodG formation in HL-60 cells but not in its H 2O 2-resistant clone HP 100 cells. o-Phenanthroline significantly inhibited the 8-oxodG formation in HL-60 cells, confirming the involvement of metal ions in the 8-oxodG formation by hydroxylamine. Electron spin resonance spectroscopy, utilizing Fe[ N-(dithiocarboxy)sarcosine] 3, demonstrated that nitric oxide (NO) was generated from hydroxylamine and esterase-treated N-hydroxyurethane. It is concluded that urethane may induce carcinogenesis through oxidation and, to a lesser extent, depurination of DNA by its metabolites.

Luteotropic roles of prolactin in early pregnant hamsters.

Prolactin (PRL) has long been regarded as a luteotropin maintaining early pregnancy in rodents. To delineate luteotropic roles of PRL in terms of luteal vascularization and immune privilege, luteal expression of Thy-1 differentiation protein, Fas, and Fas ligand (FasL) in early pregnancy was studied in hamsters on Day 4 of pregnancy (P4 group). Release of pituitary PRL was blocked by daily treatment with bromocriptine (1 mg s.c. given at 1000 h) on Days 1-4 of pregnancy (PB group). PRL withdrawal induced functional luteolysis, as evidenced by a precipitous drop in serum progesterone to background levels. In situ 3' end-labeling of fragmented DNA (TUNEL method) also clearly showed that many apoptotic nuclei accumulated in the disintegrated luteal vessels in the corpus luteum in the PB group. Immunohistochemical studies showed that luteal Thy-1-positive vascular pericytes were abundant in the P4 group but rare in the PB group. Thus, PRL is essential for luteal vascularization in early pregnancy. Western blotting and quantitative real-time reverse transcription-polymerase chain reaction data showed that Fas protein and mRNA levels increased, whereas those of FasL decreased after PRL withdrawal. Accordingly, apoptosis initiated by Fas-FasL interaction is involved in the bromocriptine-induced luteolysis. Therefore, luteotropic roles of PRL are to support Thy-1 positive pericytes in maintaining proper luteal vascularization and to prevent immune insult by preserving a balance between luteal Fas and FasL expression in early pregnancy.

Dynamic coupling of 99mTc-MIBI efflux and apoptotic pathway activation in untreated breast cancer patients.

Our previous studies showed that the efflux rate of technetium-99m methoxyisobutylisonitrile (MIBI) is directly correlated to P-glycoprotein (Pgp) levels in breast carcinoma. The aim of this study was to test whether the Pgp-dependent efflux of 99mTc-MIBI is related to the apoptotic pathway activation in breast carcinoma. Thirty-three untreated non-consecutive patients were intravenously injected with 740 MBq 99mTc-MIBI and serial images were obtained up to 4 h. The rate of efflux was determined by mono-exponential fitting of decay-corrected time-activity curves. Tumour specimens were then obtained at surgery and processed for the determination of the apoptotic index by in situ end-labelling of DNA fragments (Tunel). The rate of tumour cell proliferation was also determined using Ki67 monoclonal antibody. All breast carcinomas showed focal uptake of 99mTc-MIBI and the time to half clearance varied between 85 and 574 min. The apoptotic index ranged between 0.3% and 4.2%, whereas the rate of proliferation varied between 13% and 40%. We found a positive and significant correlation between the apoptotic index and the rate of proliferation (r=0.79, P<0.0001). The efflux rate of 99mTc-MIBI was directly and significantly correlated with the apoptotic index (r=0.74, P<0.0001) and with the rate of proliferation (r=0.58, P<0.001). After partial correlation analysis, only the apoptotic index showed a significant correlation with the efflux rate of 99mTc-MIBI (r=0.57, P<0.001). Our findings indicate that enhanced transport activity of Pgp is associated with increased activation of the apoptotic pathway, suggesting that inhibition of Pgp function with specific modulators may be effective in these patients. Furthermore, since mitochondria are central executioners of apoptosis and intracellular sites of 99mTc-MIBI sequestration, a model for the dynamic coupling of Pgp-dependent 99mTc-MIBI efflux and apoptotic pathway activation may be derived.

Glucocorticoids and progesterone prevent apoptosis in the lactating rat mammary gland.

Apoptosis, a form of noninflammatory cell death, plays a central role in mammary gland involution after weaning. Previous studies have shown that apoptosis in the postweaning mammary gland is substantially reduced by treatment with glucocorticoids or progesterone, but whether these steroids exert a similar antiapoptotic effect during normal lactation is not known. Therefore, the present study used an in vivo rat model to assess the effects of progesterone and glucocorticoids on apoptosis in the lactating mammary gland. Rats were untreated, sham operated, ovariectomized (OVX), and/or adrenalectomized (ADX) on d 10 of lactation. Additional groups of OVX/ADX rats were treated with either progesterone or corticosterone. Mammary gland apoptosis was determined 3 d later by 3'-end labeling of fragmented DNA and by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling analysis (TUNEL). DNA fragmentation was relatively low in the mammary gland from untreated and sham-operated rats and was unaffected by either ADX or OVX alone. In contrast, DNA fragmentation was markedly elevated in OVX/ADX rats (P < 0.01), but this effect on mammary gland apoptosis was prevented by replacement with either corticosterone or progesterone. Consistent with these data, dying cells identified by TUNEL analysis were readily observed in the alveolar epithelium of mammary tissue from OVX/ADX rats but not in any of the other groups. These data demonstrate that during normal lactation, mammary gland apoptosis is inhibited by endogenous progesterone and glucocorticoids. Importantly, the presence of either steroid alone was sufficient to prevent apoptosis, suggesting that their antiapoptotic effects in the lactating mammary gland may be mediated via similar signaling pathways.

Cytoplasmic c-Jun N-terminal immunoreactivity: a hallmark of retinal apoptosis.

1. We investigated the association of c-Jun with apoptosis within retinal tissue. Explants of the retina of neonatal rats were subject to a variety of procedures that cause apoptosis of specific classes of retinal cells at distinct stages of differentiation. The expression of c-Jun was detected by Western Blot, and immunohistochemistry was done with antibodies made for either N-terminal or C-terminal domains of c-Jun, and correlated with apoptosis detected either by chromatin condensation or by in situ nick end labeling of fragmented DNA. 2. c-Jun protein content was increased in retinal tissue subject to induction of both photoreceptor and ganglion cell death. 3. c-Jun N-terminal immunoreactivity was found mainly in the cytoplasm of apoptotic cells regardless of cell type, of the stage of differentiation, including proliferating cells, or of the means of induction of apoptosis. 4. The data are consistent with the hypothesis that c-Jun is involved in the control of cell death in retinal tissue, but other proteins that cross-react with c-Jun N-terminal antibodies may also be major markers of retinal apoptosis. 5. Antibodies directed to c-Jun N-terminal (aa 91-105) are useful tools to follow apoptotic changes in retinal tissue.

Apoptotic cell death in suspension cultures of Taxus chinensis var. mairei

Apoptotic cell death was observed in suspension cultures of Taxus chinensis var. mairei under normal cultivation conditions by using microscopy, total DNA agarose gel electrophoresis and in situ end-labeling of fragmented DNA. The morphological and biochemical changes of cells occurred mainly in the non-dividing cell clusters, indicating that the T. chinensis cells died mainly by apoptosis. There exists a close relationship between cell apoptosis and Taxol formation. Taxol concentration increased with the increase in content of apoptotic cells and reached a maximum (14.2 mg l−1) after 23 days of culture, corresponding to a maximum ratio of apoptotic to total cells of about 13%.