Interferon-alpha in chronic hepatitis C.
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Antiviral Agents↗
Biomedical subjects
S Bruno
Publications and source records attributed to S Bruno.
Dynamics of hypervariable region 1 variation in hepatitis C virus infection and correlation with clinical and virological features of liver disease.
Hepatitis C virus (HCV) infection is a dynamic process during which molecular variants are continuously selected as the result of virus adaptation to the host. Understanding the nature of HCV genetic variation is central to current theories of pathogenesis and immune response. We prospectively studied hypervariable region 1 (HVR1) variation in the E2 gene of 36 hepatitis C patients, including 10 asymptomatic carriers, followed up for 1 to 2 years. Sequence changes in single and consecutive serum samples were assessed and correlated with clinical and virological parameters of liver disease. A region of the E1 gene was sequenced for comparison in 3 subjects. HVR1 heterogeneity at single time points widely varied in individual patients, did not increase cumulatively over the follow-up period, and did not correlate with HVR1 evolutionary rates. Conversely, the process of HVR1 sequence diversification, although differed considerably among patients, was stable over time and directly correlated with infections by HCV type 2, lower alanine aminotransferase (ALT) levels, and absence of cirrhosis. HCV carriers showed the highest HVR1 variation rates. Our findings indicate that HVR1 variation has an adaptive significance and is associated with favorable features of liver disease and suggest that prospective, rather than static, observations are required to model the process of HCV variation.
Adult↗
Modulation of the insulin-like growth factor-I system by N-(4-hydroxyphenyl)-retinamide in human breast cancer cell lines.
The potent mitogenic activity of insulin-like growth factor I (IGF-I) on breast epithelium is inhibited by retinoic acid in oestrogen receptor-positive (ER+) breast cancer cell lines. We studied and compared the effects of N-(4-hydroxyphenyl)-retinamide (4-HPR) in terms of growth inhibition and modulation of the IGF-I system in ER+ (MCF-7) and oestrogen receptor-negative (ER-) (MDA-MB231) breast cancer cell lines. Treatment with 1-10 microM 4-HPR for up to 96 h induced a dose- and time-dependent inhibition of proliferation in both breast cancer cell lines. Induction of apoptosis was much more evident in MCF-7 than in MDA-MB231 cells (30-40% compared with 0-5% respectively at 5 microM for 48 h). Exogenous human recombinant IGF-I (hr-IGF-I)-stimulated cell proliferation was abolished by 1 microM 4-HPR in MCF-7 cells. Immunoreactive IGF-I-like protein concentration in conditioned medium was reduced by 38% in MCF-7 and by 90% in MDA-MB231 cell lines following treatment for 48 h with 5 microM 4-HPR. Western ligand blot analysis showed a reduction of IGF-binding protein 4 (BP4) and BP5 by 67% and 87%, respectively, in MCF-7, whereas IGF-BP4 and -BP1 were reduced by approximately 20% in MDA-MB231 cells. Exposure to 5 microM 4-HPR for 48 h inhibited [125I]IGF-I binding and Scatchard analysis revealed a decrease of more than 50% in maximum binding capacity (Bmax) and a reduced receptor number/cell in both cancer cell lines. Steady-state type I IGF-receptor mRNA levels were reduced by approximately 30% in both tumour cell lines. We conclude that 4-HPR induces a significant down-regulation of the IGF-I system in both ER+ (MCF-7) and ER- (MDA-MB231) breast cancer cell lines. These findings suggest that, in our model, interference with the ER signalling pathway is not the only mechanism of breast cancer growth inhibition by 4-HPR.
Antineoplastic Agents↗
Hepatocyte proliferation rate is a powerful parameter for predicting hepatocellular carcinoma development in liver cirrhosis.
AIMS: A sound predictive test is lacking for the identification of cirrhotic patients at high risk of developing hepatocellular carcinoma. The present study evaluates the measurement of hepatocyte expression of silver stained nucleolar organiser region (AgNOR) proteins as a risk factor for the development of hepatocellular carcinoma in cirrhosis. METHODS: Liver biopsies from 176 cirrhotic patients included in a follow up surveillance programme for hepatocellular carcinoma development were evaluated prospectively for hepatocyte AgNOR protein quantity. The follow up programme consisted of clinical and biochemical assessment every three months, and ultrasound scanning and serum alpha-fetoprotein (alpha FP) assessment every six months. Histological sections from the needle biopsies performed at enrollment were stained selectively for AgNOR proteins and the percentage of hepatocytes with an AgNOR protein area > or = 7 micron 2, indicative of a proliferative state (AgNOR proliferation index (AgNOR-PI)), was measured. RESULTS: During the mean (SD) follow up time of 65.5 (36.29) months (range, 12-143; median, 67), hepatocellular carcinoma was diagnosed in 48 of 176 patients (27.3%). The AgNOR-PI of the whole series ranged from 0% to 5% (median, 0.9%), and was significantly higher in patients with liver cell dysplasia and hepatitis B surface antigen (HBsAg) positivity (p < 0.0001 and p = 0.0002, respectively). The 176 patients were divided into two groups according to their AgNOR-PI scores; a cut off value of 2.5% defined by the receiver operating characteristic curve and the Youden index was used. Forty two patients were included in the high AgNOR-PI (< 2.5%) group, and 134 patients the low AgNOR-PI (< 2.5%) group. In the high AgNOR-PI group, 25 of 42 patients developed hepatocellular carcinoma, in contrast to only 23 of 134 patients (17.2%) in the group with a low AgNOR-PI (p < 0.0001). Hepatocellular carcinoma development was also significantly more frequent in patients with liver cell dysplasia and HBsAg positivity. Multivariate analysis using AgNOR-PI, liver cell dysplasia, HBsAg positivity, and hepatitis C virus (HCV) infection as covariates demonstrated that the AgNOR-PI parameter was the only significant predictor of hepatocellular carcinoma development. CONCLUSIONS: These results demonstrate that a high hepatocyte proliferation rate is a major risk factor for hepatocellular carcinoma development in the cirrhotic liver. Therefore, the evaluation of the hepatocyte proliferation rate is very important to identify patients requiring a more strict follow up programme for early diagnosis of hepatocellular carcinoma.
Adult↗
Clinical significance of hepatic HCV RNA in patients with chronic hepatitis C demonstrating long-term sustained response to interferon-alpha therapy.
Whether sustained biochemical response and absence of serum HCV RNA in the 6-12 months following suspension of interferon-alpha (IFN-alpha) therapy reflect definitive viral clearance in patients with chronic hepatitis C virus (HCV) infection is controversial. To obtain more information on this topic, HCV RNA was sought in both liver and serum samples of 25 long-term responders who were followed for a median period of 39 months (range 21-79) after discontinuation of IFN-alpha. Liver biopsy was undertaken before and 6 to 12 months after IFN-alpha withdrawal. Liver and serum HCV RNA were tested by a nested polymerase chain reaction. Twenty-two patients (88%) tested negative for both liver and serum HCV RNA, two patients had detectable HCV RNA in both liver and serum, and one patient showed persistent HCV RNA only in the liver. Post-treatment liver histology improved markedly in all patients, including those with viral persistence. During further follow-up, biochemical remission was maintained in all patients except one in whom both serum and liver specimens remained HCV RNA positive. The data indicate that the large majority of long-term responders test negative for HCV RNA in the liver, which suggests definitive eradication of HCV RNA infection.
Adolescent↗
Hepatitis C virus genotypes and risk of hepatocellular carcinoma in cirrhosis: a prospective study.
A prospective study was performed to establish whether infection with specific hepatitis C virus (HCV) genotypes was associated with an increased risk of development of hepatocellular carcinoma (HCC) in cirrhosis. A cohort of 163 consecutive hepatitis C virus antibody (anti-HCV)-positive cirrhotic patients was prospectively evaluated for the development of HCC at 6-month intervals by ultrasound (US) scan and alpha-fetoprotein (AFP) concentration. HCV genotypes were determined according to Okamoto. Risk factors associated with cancer development were analyzed by univariate and multivariate statistics. At enrollment, 101 patients (62%) were infected with type 1b, 48 (29.5%) were infected with type 2a/c, 2 (1.2%) were infected with type 3a, 1 (0.6%) was infected with type 1a, 3 (1.8%) had a mixed-type infection, and, in 8 patients (4.9%), genotype could not be assigned. After a 5- to 7-year follow-up (median, 68 months), HCC developed in 22 of the patients, 19 infected with type 1b and 3 with type 2a/c (P < .005). Moreover, HCC developed more frequently in males (P < .01), patients with excessive alcohol intake (P < .01), those over 60 years of age (P < .02), and in patients who did not receive interferon treatment (P < .02). Multivariate analysis showed that type 1b was the most important risk factor associated with tumor development (odds ratio 6.14, 1.77-21.37 95% confidence interval). Other independent risk factors were older age and male sex. Cirrhotic patients infected with HCV type 1b carry a significantly higher risk of developing HCC than patients infected by other HCV types. The latter may require a less intensive clinical surveillance for the early detection of neoplasia.
Adult↗
Behçet's disease: an immune-mediated vasculitis involving vessels of all sizes.
Behçet's disease is an immune-mediated vasculitis affecting both small and large vessels. Small-vessel vasculitis is the pathological basis of the multiorgan involvement that results in protean clinical features. However, relapsing aphthous ulcers in the mouth are considered the clinical hallmark and are often also observed over the genitalia. Both manifestations, in association with uveitis, form the typical clinical triad. In addition, skeletal muscles, joints, gastrointestinal, cardiopulmonary, and central nervous systems can be involved. Heterogeneity in incidence, clinical manifestations, course, and severity are observed according to ethnic background. The natural course is chronic with relapses and remissions, gradually abating over the years, but the illness can also be life or sight threatening. Its origin and cause are still obscure: genetic, infectious, environmental, and immunological factors have been proposed. Owing to the lack of a specific test, diagnosis still relies on recognition of the typical clinical pattern. Treatment usually includes corticosteroids and immunosuppressive drugs. A better understanding of the pathogenesis will hopefully improve both diagnosis and therapy. In addition, the development of tests aimed at monitoring disease activity and response to therapy is certainly desirable.
Behcet Syndrome↗
Quantitative analysis of hepatitis C virus activity in vivo in different groups of untreated patients.
Highly sensitive competitive PCR (cPCR) and competitive reverse transcription PCR (cRT-PCR) methodologies were recently developed and applied for quantifying viral DNA and RNA species (including HCV RNA) present in clinical samples at low concentration. In this study, we used cRT-PCR to compare the viral load of 118 untreated patients with HCV infection and different clinical conditions (80 patients with chronic hepatitis, 18 infected subjects with persistently normal ALT levels and various degrees of liver injury, 10 HCV infected subjects that tested positive for anti-LKM1 antibodies, and 10 patients with HCV infection and cryoglobulinemia). The results indicate that while great individual variability of HCV viremia is detectable even among patients with similar clinical conditions, the mean HCV RNA copy number in samples from patients with different clinical conditions was similar in all groups with the single exception of patients that tested positive for anti-liver-kidney microsomal auto-antibodies type 1 (anti-LKM1); interestingly, lower HCV viremia levels were revealed in these anti-LKM1-positive cases with liver disease of uncertain pathogenesis.
Gene Dosage↗
Ultrasonography-detected macroregenerative nodules in cirrhosis: a prospective study.
BACKGROUND & AIMS: The preneoplastic role of macroregenerative nodules in cirrhosis is still debated. Thirty-two consecutive ultrasonography-detected macronodules were followed up to evaluate whether and which lesions are the actual precursors of hepatocellular carcinoma, if histology can identify preneoplastic macronodules, and whether additional parameters are related to neoplastic evolution. METHODS: Macroregenerative nodule classification was based on recently proposed histological criteria. Extranodular liver cell dysplasia was also evaluated. The follow-up included ultrasonography and serum alpha-fetoprotein level determination every 3 months. RESULTS: Twenty-two macronodules (78%) were classified as typical and 7 (22%) as atypical. Twenty-one were hypoechoic, and 11 were hyperechoic. Extranodular dysplasia was more frequently associated with atypical than typical macronodules (5 of 7 vs. 6 of 22). After 28 +/- 15 months, neoplastic transformation occurred in 8 macronodules (25%) and was more frequent in atypical than in typical (5 of 7 vs. 3 of 25), in hyperechoic than in hypoechoic (5 of 11 vs. 3 of 21), and in extranodular dysplasia-associated macronodules than in extranodular dysplasia-free macronodules (5 of 11 vs. 2 of 18). Five hepatocellular carcinomas appeared outside the original macronodule. CONCLUSIONS: Atypical macroregenerative nodules can be considered precursors of hepatocellular carcinoma. Histology is useful in identifying preneoplastic macronodules, and hyperechoic pattern and extranodular dysplasia are additional risk factors for neoplastic transformation.
Aged↗
Variable clinical phenotype in merosin-deficient congenital muscular dystrophy associated with differential immunolabelling of two fragments of the laminin alpha 2 chain.
Approximately half the cases of classical congenital muscular dystrophy (CMD) have a pronounced deficiency or absence of the laminin alpha 2 chain of laminin-2 (merosin). This is caused by mutations in the LAMA2 gene that codes for laminin alpha 2, and all informative cases so far studied show linkage to the appropriate region on chromosome 6q. Most CMD patients with a deficiency of laminin alpha 2 have a severe phenotype that involves skeletal muscle, and the central and peripheral nervous system. We have identified four cases that have minimal reduction of laminin alpha 2 using a commercial antibody that only recognises a C-terminal 80 kDa fragment, but show a pronounced reduction using an antibody to the 300 kDa fragment. Haplotype analysis is compatible with linkage to the LAMA2 locus in three informative families, whilst the fourth family was not informative. Two of the affected children are ambulant and have a mild phenotype. The third case is unusual in having severe muscle weakness but does not show the white matter changes on magnetic resonance imaging of the brain that is usually seen in merosin-deficient cases of CMD; the fourth case has a severe phenotype, typical of merosin-deficient patients but shows good immunolabelling of the 80 kDa fragment of laminin alpha 2, corresponding to the C-terminal region. Our data show that there is a broad spectrum of phenotype and protein expression associated with a primary deficiency in laminin alpha 2, and that a wider range of clinical cases need to be screened for a deficiency of merosin. It is also important to study the expression of laminin alpha 2 with more than one antibody.
Antibody Specificity↗
Expression of laminin chains in skin in merosin-deficient congenital muscular dystrophy.
Laminin-2 (merosin) is a heterotrimer composed of alpha 2, beta 1 and gamma 1 chains. Approximately half of the cases with the classical form of congenital muscular dystrophy (CMD) have a deficiency of the laminin alpha 2 chain, encoded by the LAMA2 gene on chromosome 6q22. This disorder is often termed merosin-deficient CMD. Skeletal and cardiac muscle, and the peripheral and central nervous systems, all express laminin alpha 2 and can be affected in merosin-deficient CMD. Normal skin also expresses all three chains of laminin-2 at the epidermal/dermal junction, around hair follicles and in the sensory nerves. Skin biopsies can therefore be used to assess merosin status in patients. We show here an absence of laminin alpha 2 in skin from four cases of CMD with a severe phenotype and abnormal magnetic resonance image (MRI) of the brain, in contrast to normal expression in one case of mild CMD with normal MRI, and in five controls. An additional case of CMD had a partial deficiency of laminin alpha 2 in the skin and severe motor disability, but a normal MRI. Sensory nerves in this case showed normal expression of laminin alpha 2, in contrast to its absence in the severe cases. The expression of laminin beta 1 was also reduced in skin from cases of merosin-deficient CMD. In contrast to human fetal muscle, the laminin alpha 2 protein was not detected in fetal skin up to 23 weeks of gestation. The laminin beta 1 and gamma 1 chains, and the mRNA for laminin alpha 2, however, were present. Studies of mRNA of cultured skin cells suggest that fibroblasts are the major source of laminin alpha 2, not keratinocytes. Our data show that skin is useful for the assessment of merosin status in patients with CMD and that skin fibroblasts may be a useful source of tissue-specific RNA. In addition, we show that there is a tissue-specific difference in the developmental expression of the laminin alpha 2 protein.
Adolescent↗
Minimal immunological effects on workers with prolonged low exposure to inorganic mercury.
OBJECTIVES: This study was carried out to investigate possible immunological changes in workers with prolonged low exposure to inorganic mercury in a fluorescent light bulb factory. METHODS: 29 immunological variables were examined in 34 workers with prolonged low level exposure to inorganic mercury (exposed workers) and 35 unexposed workers as the controls. The selected indicator of mercury exposure was concentration of mercury in the urine (U-Hg), which declined progressively from 36.0 micrograms/l in 1978 to 6.0 micrograms/l in the study year 1994. RESULTS: None of the exposed workers had ever shown signs of either acute or chronic inorganic mercury toxicity or had shown any form of hypersensitivity. The only changes found in the exposed workers, compared with the controls, were a reduction of the cells that express cluster differentiation (CD25,(T activation antigen (Tac antigen))) and concentrations of tumour necrosis factor-alpha (TNF-alpha) in serum. However, the decrease of cells that express CD25 was unrelated to occupational exposure and was, in all likelihood a chance finding. Conversely, the decline in serum TNF-alpha was closely associated with occupational exposure. However, no dose-response relation was found between U-Hg and TNF-alpha concentrations; nor were TNF-alpha concentrations affected by cumulative occupational exposure to inorganic mercury in over 20 years. CONCLUSIONS: Tentatively, we suggest that reduced serum TNF-alpha concentrations might be indicative of an in vivo functional defect of the monocyte macrophage system in this particular group of workers even though they were clinically asymptomatic.
Biomarkers↗
Randomized trial of CVPP for three versus six cycles in favorable-prognosis and CVPP versus AOPE plus radiotherapy in intermediate-prognosis untreated Hodgkin's disease.
PURPOSE: To evaluate in a randomized trial the impact of three versus six cycles of cyclophosphamide, vinblastine, procarbazine, and prednisone (CVPP) chemotherapy in favorable-prognosis and CVPP versus doxorubicin, vincristine, prednisone, and etoposide (AOPE) plus involved-field radiotherapy (RT) in intermediate-prognosis previously untreated Hodgkin's disease. PATIENTS AND METHODS: Of 256 patients evaluated, 80 with a favorable prognosis according to a prognostic index designed by the Grupo Argentina de Tratamiento de Leucemia Aguda (GATLA) were randomized to three versus six cycles of CVPP without RT and 176 with intermediate risk to CVPP versus AOPE, both for six cycles with RT between the third and fourth cycles of 30 Gy to the involved areas at diagnosis. CVPP consisted of intravenous (I.V.) cyclophosphamide and vinblastine on days 1 and 8, and oral procarbazine and prednisone on days 1 to 14, every 28 days. AOPE consisted of I.V. doxorubicin and vincristine on day 1, oral prednisone on days 1 to 5, and I.V. etoposide on days 1 and 3, every 28 days. RESULTS: Complete remission was obtained in 39 of 41 (95%) patients treated with three cycles of CVPP and 36 of 39 (92%) treated with six cycles in the favorable-risk group (difference not significant [NS]). In the intermediate-risk group, 89 of 92 (97%) treated with CVPP plus RT versus 75 of 84 (89%) treated with AOPE plus RT achieved a complete remission (P = .05). At 60 months, the event-free survival (EFS) and overall survival rates in the favorable-risk group were 80% and 91% for CVPP x 3 and 84% and 97% for CVPP x 6, respectively (P = NS). In the intermediate-risk group, 60-month EFS rate for CVPP plus RT was 85%, compared with 66% for AOPE plus RT (P = .009). The overall survival rate was 95% versus 87% respectively (P = .157). CONCLUSION: Three cycles of CVPP without RT are equally effective as six cycles in the favorable-risk group. However, in the intermediate-group, CVPP plus RT is superior to AOPE plus RT, with significantly fewer events before and after induction (P = .009), without a difference in overall survival.
Adolescent↗
Histological changes but not virus eradication reflect biochemical improvement in chronic hepatitis C patients showing a long-term response to interferon alpha therapy.
AIMS: To assess the relationship between changes in liver histology and virological parameters of HCV infection in patients with a sustained biochemical response to alpha-interferon treatment for chronic hepatitis, with the aim of identifying the most reliable indicator of treatment efficacy. METHODS: Quantitative serial testing of HCV-RNA in plasma samples collected on a monthly basis over the last period of post-treatment follow-up were tested in sixteen subjects with > or = 12 months aminotransferase normalisation following discontinuation of interferon therapy. The quantitative HCV-RNA determination in plasma was performed by a competitive reverse-transcription polymerase-chain reaction. Pretreatment and 12-month post-treatment liver histologies were blindly evaluated using a semi-quantitative scoring system. At these times a qualitative HCV-RNA analysis was also carried out. RESULTS: The post-treatment histological grading score was significantly reduced. Nevertheless, all patients except one tested positive for HCV-RNA in plasma (mean 1.7 x 10(6) molecules/ml): nine were positive in all the serial determinations, while the remaining 6 were intermittently positive. No relationship between genotype, viral load, pattern of viremia (intermittent or continuous) and change in histological score was found. CONCLUSIONS: Changes in liver histology are the most reliable indicator of the efficacy of interferon treatment in hepatitis C related liver disease. HCV-RNA clearance based on serial sampling does not appear to be a reliable indicator, even in the presence of a sustained biochemical response and histological improvement.
Adult↗
Eosinophilic ileocolitis by Enterobius vermicularis: a description of two rare cases.
BACKGROUND: Human enterobiasis is usually mild. Occasionally, however, an "ectopic" disease may occur with a more severe course. Two rare cases of eosinophilic ileocolitis due to Enterobius vermicularis infection are reported here. CASE REPORTS: Case n degree 1 was 46 years old, presenting with fever and bloody diarrhoea. Blood eosinophilia was present. Stool microscopy demonstrated red blood cells and leukocytes. A 2 mm long worm with bilateral cervical wings was found in wet-mount preparations of faecal samples. The Scotch tape test was positive for Enterobius vermicularis eggs. Colon biopsy specimens showed massive eosinophilic infiltration and a typical pinworm section overlying the infiltrated mucosa. Case n degree 2 was a 24-year-old, anti-HIV negative homosexual, presenting with watery diarrhoea. Tests for malabsorption were negative. Three mm long adult male E. vermicularis were found on stool microscopy. Biopsy specimens from the colon showed eosinophilic infiltration. In both cases a 200 mg/day course of oral mebendazole eliminated the symptoms within 3 days. CONCLUSIONS: In these two cases the clinical presentation of enterobiasis was atypical. A common finding was the eosinophilic infiltration of bowel mucosa, although it is still uncertain whether the worm per se may induce mast cell degranulation and eosinophil activation. Nevertheless, the possibility of Enterobius vermicularis infection should be considered in the presence of eosinophilic ileocolitis.
Adult↗
The mouse region syntenic for human spinal muscular atrophy lies within the Lgn1 critical interval and contains multiple copies of Naip exon 5.
Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval for Lgn1, a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacterium Legionella pneumophila. We have generated a mouse YAC contig across the Lgn1/Sma interval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to the Lgn1 critical region, making these two genes candidates for the Lgn1 phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy of Naip exon 5. Identification of the Lgn1 gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.
Animals↗
The cell cycle effects of camptothecin.
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Animals↗
Effect of protease inhibitors on early events of apoptosis.
Proteolysis is an early event of apoptosis which appears to be associated with activation of the endonuclease which is responsible for internucleosomal DNA cleavage. The present study was designed to reveal the possible role of proteolysis in other early events, such as chromatin condensation, nuclear breakdown, and destabilization of in situ DNA double-stranded structure. Apoptosis of human leukemic HL-60 cells and rat thymocytes was induced by different agents, including DNA topoisomerase inhibitors, an RNA antimetabolite, and the glucocorticosteroid, prednisolone. DNA degradation was evaluated by pulsed field and conventional gel electrophoresis and by the presence of in situ DNA strand breaks. DNA stability was estimated by the measure of its sensitivity in situ to denaturation. Chromatin condensation, nuclear breakdown, and other morphological changes were monitored by interference contrast and UV microscopy following cell staining with the DNA-specific fluorochrome 4',6-diamidino-2- phenylindole. Several irreversible or reversible serine protease inhibitors prevented internucleosomal DNA degradation, nuclear breakdown, and destabilization of DNA double-stranded structure. The effective inhibitors, however, did not prevent the onset of chromatin condensation, nor the loss of the fine structural framework, nor the initial step of DNA cleavage generating DNA fragments of >=50 kb in size. The data indicate that in both cell systems the activity of proteases sensitive to the inhibitors tested is needed for internucleosomal DNA cleavage to occur. The data also suggest that these proteases may be involved in dissolution of the nuclear envelope. Because nuclear matrix proteins and histones stabilize DNA in situ, and the decrease in DNA stability which occurs during apoptosis is precluded by the inhibitors, it is likely that serine proteases may degrade DNA stabilizing proteins. The activity of these proteases, however, appears needed neither for DNA cleavage to >=50-kb fragments nor for the onset of chromatin condensation which is associated with dissolution of the structural framework of the nucleus.
Animals↗