Proteins in Outer Membrane Vesicles Produced by Burkholderia cepacia are Responsible for Pro-inflammatory Responses in Epithelial Cells

Gram-negative bacterial pathogens produce outer membrane vesicles (OMVs) and this secreted cargo plays a role in host-pathogen interactions. OMVs isolated from Burkholderia cepacia induce the cytotoxicity and pro-inflammatory responses both in vitro and in vivo, but OMV components associated with host pathology have not been characterized. This study analyzed the proteomes of OMVs produced by B. cepacia ATCC 25416 and investigated whether proteins in B. cepacia OMVs were responsible for host pathology in vitro. Proteomic analysis revealed that a total of 265 proteins were identified in B. cepacia OMVs. Of the 265 OMV proteins, 179 (67.5%), 32 (12.1%), 27 (10.2%), 17 (6.4%), and 10 (3.8%) were predicted to be located in the cytoplasm, inner membrane, periplasmic space, outer membrane, and extracellular compartment, respectively. Several putative virulence factors were also identified in B. cepacia OMVs. B. cepacia OMVs slightly induced the cytotoxicity in lung epithelial A549 cells, but there was no difference in cytotoxic activity between intact OMVs and proteinase K-treated OMVs. B. cepacia OMVs stimulated the expression of pro-inflammatory cytokine and chemokine genes in A549 cells, but the expression of these cytokine genes was significantly inhibited in A549 cells incubated with proteinase K-treated OMVs. In conclusion, our results suggest that proteins in B. cepaciaOMVs are directly responsible for pro-inflammatory responses in lung epithelial cells.

Proteins in Outer Membrane Vesicles Produced by Burkholderia cepacia are Responsible for Pro-inflammatory Responses in Epithelial Cells

Gram-negative bacterial pathogens produce outer membrane vesicles (OMVs) and this secreted cargo plays a role in host-pathogen interactions. OMVs isolated from Burkholderia cepacia induce the cytotoxicity and pro-inflammatory responses both in vitro and in vivo, but OMV components associated with host pathology have not been characterized. This study analyzed the proteomes of OMVs produced by B. cepacia ATCC 25416 and investigated whether proteins in B. cepacia OMVs were responsible for host pathology in vitro. Proteomic analysis revealed that a total of 265 proteins were identified in B. cepacia OMVs. Of the 265 OMV proteins, 179 (67.5%), 32 (12.1%), 27 (10.2%), 17 (6.4%), and 10 (3.8%) were predicted to be located in the cytoplasm, inner membrane, periplasmic space, outer membrane, and extracellular compartment, respectively. Several putative virulence factors were also identified in B. cepacia OMVs. B. cepacia OMVs slightly induced the cytotoxicity in lung epithelial A549 cells, but there was no difference in cytotoxic activity between intact OMVs and proteinase K-treated OMVs. B. cepacia OMVs stimulated the expression of pro-inflammatory cytokine and chemokine genes in A549 cells, but the expression of these cytokine genes was significantly inhibited in A549 cells incubated with proteinase K-treated OMVs. In conclusion, our results suggest that proteins in B. cepaciaOMVs are directly responsible for pro-inflammatory responses in lung epithelial cells.

Clinical Features of Staphylococcal Scalded Skin Syndrome Caused by Community-Associated Methicillin-Resistant Staphylococcus aureus in Changwon City, Korea, during 2006 and 2015

PURPOSE: We investigated the clinical features and epidemiology of staphylococcal scalded skin syndrome (SSSS) from year 2006 to 2015 in Changwon city, Korea. METHODS: We reviewed medical records of 69 patients diagnosed with SSSS from year 2006 to 2015. Antibiotic susceptibility testing was performed by agar dilution method. Methicillin-resistant Staphylococcus aureus (MRSA) was phenotypically identified by oxacillin susceptibility testing and genotypically confirmed by the existence of the mecA gene. RESULTS: The median age of patients was 2.0 years (range 0.2–6 years). Three (4.3%), 53 (76.8%), and 13 (18.9%) patients showed the generalized type, the intermediate type, and the abortive type, respectively. Patients occurred throughout the year, but most patients occurred between July and October. MRSA was isolated from 54 of the 60 patients regardless of the clinical types. All patients recovered without any complications. CONCLUSIONS: There was a constant occurrence of SSSS patients caused by MRSA in Changwon area during 2006 and 2015. It is needed to constantly monitor the occurrence of patients with SSSS.

Antimicrobial Susceptibility and Clonal Distribution of the Blood Isolates of Pseudomonas aeruginosa from Two Korean Hospitals

An increasing prevalence of infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) causes a serious therapeutic problem in clinical setting. This study investigated the antimicrobial susceptibility, resistance mechanisms against aminoglycosides, and molecular epidemiology of 76 blood isolates of P. aeruginosa from two Korean hospitals. Thirty-four isolates were susceptible to all 13 antimicrobial agents tested, whereas 28 isolates showed a MDR or extensively drug-resistant phenotype. There was a significant difference in resistance rates of P. aeruginosa isolates against aztreonam, piperacillin-tazobactam, imipenem, meropenem, ciprofloxacin, and norfloxacin between two hospitals. Genes for aminoglycoside-modifying enzymes (AMEs), including aphA6 (n = 14), aadB (n = 11), aacA4 (n = 8), and aphA1 (n = 1), and 16S rRNA methylase armA (n = 6) were detected in 26 P. aeruginosa isolates resistant to aminoglycosides. There was no significant difference in carriage of genes for AME and 16S rRNA methylase between two hospitals, but aacA4 and aphA1 were specifically detected in P. aeruginosa isolates from one hospital. Seventy-six P. aeruginosa isolates were classified into 55 pulsotypes at similarity value of 0.85, and 31 and 24 pulsotypes were specifically detected in each hospital. This study demonstrates that differences in antimicrobial susceptibility of P. aeruginosa isolates between two hospitals are possibly due to the presence of diverse clones specific in each hospital.

Prediction of Nuclear Targeting Proteins with Nuclear Localization Signals in Staphylococcus aureus and Nuclear Targeting of beta-lactamase in Host Cells

Nuclear targeting of bacterial proteins in host cells and subsequent interaction with nuclear molecules are an emerging pathogenic mechanism of bacteria. In this study, we predicted the nuclear targeting proteins with nuclear localization signals (NLSs) in Staphylococcus aureus using bioinformatic analysis. A total of 51 proteins of S. aureus, comprising of 24 functional and 27 hypothetical proteins, were predicted to carry putative NLSs. Among them, beta-lactamase and MsrR proteins with the putative NLSs were selected to determine the nuclear targeting in host cells. Fusion proteins of BlaZ-green fluorescent protein (GFP) were evenly distributed in the nuclei of host cells and subsequently induced host cell death. However, fusion proteins of MsrR-GFP were not localized in the nuclei of host cells In conclusion, screening of nuclear targeting proteins with NLSs and determination of their pathology in host cells may open up the new field of S. aureus pathogenesis.

Staphylococcus aureus Membrane Vesicles and Its Potential Role in Bacterial Pathogenesis

The production of extracellular vesicles is a ubiquitous process in both Gram-negative and Gram-positive bacteria. Gram-negative bacteria produce and secrete outer membrane vesicles during in vitro culture and in vivo infection and their contribution to bacterial pathogenesis has been well characterized. However, little is known about extracellular vesicles in Gram-positive bacteria. Until now, only few Gram-positive bacterial species, Staphylococcus aureus, Bacillus anthracis, B. cereus, and B. subtilis, have been found to produce membrane vesicles (MVs), but their contribution to bacterial pathogenesis has not been understood. Here, I discuss S. aureus MVs in terms of MV production, interaction of MVs with host cells, and immune response against MVs to understand its potential role in S. aureus pathogenesis.

Distribution of Genomic Species and Antimicrobial Susceptibility in Acinetobacters Isolated from Gangjin Bay, Korea

A total of 90 Acinetobacter isolates from freshwater and seawater in Gangjin Bay of Korea was investigated for the distribution of genomic species, antimicrobial resistance patterns and clonal relatedness. By amplified ribosomal DNA restriction analysis, eighty-nine Acinetobacter isolates were classified into 11 Acinetobacter genomic species. A. johnsonii (n=23) was the most prevalent, followed by A. baumannii (n=13), A. calcoaceticus (n=13), Acinetobacter genomic species 11 (n=10), A. phenon 6/ct13TU (n=9), A. junii (n=5), A. venetianus (n=5), Acinetobacter genomic species 17 (n=4), 14BJ (n=3), A. phenon 10/1271 (n=2), Acinetobacter genomic species 3 (n=1), and ungrouped (n=1). The majority of Acinetobacter genomic species were isolated from the site A and B, and some known nosocomial pathogens in the clinical environment were observed among them. Of the 11 antimicrobial drugs tested, several A. johnsonii isolates exhibited high-frequency resistance to a wide variety of antimicrobial agents, including ampicillin-sulbactam, piperacillin, ceftazidime, cefotaxime, and sulfamethoxazole (p < 0.001). Some Acinetobacter genomic species were resistant to currently used antibiotics but all isolates were susceptible to imipenem, amikacin, and tetracycline. Based on the results of antimicrobial resistance pattern and phylogenetic analysis, 23 A. johnsonii isolates were classified into 19 pulsotypes. In conclusion, there was a significant difference in the distribution of Acinetobacter species between freshwater and seawater. Predominance of A. johnsonii strains was probably due to their ability to proliferate in the contaminated aquatic environment originated from local geographic features. Therefore, the waste effluent from animals and humans plays an important role in the distribution of Acinetobacter species in aquatic environment.

Fatal melioidosis in a tourist returning from Cambodia / 대한내과학회지

Melioidosis, which is infection with the Gram-negative bacterium Burkholderia pseudomallei, is an important cause of sepsis in Southeast Asia and northern Australia and mainly affects diabetics who come into direct contact with wet soil. It presents as a febrile illness, ranging from an acute fulminant septicemia to a chronic debilitating localized infection. Only two cases of chronic infection have been reported in Korea. Both patients had lived in Southeast Asia for more than 1 year. We report a case of melioidosis presenting as acute fulminant septicemia and pneumonia in a 47-year-old diabetic male who had visited Cambodia for 4 days, 1 month before admission. He died of refractory septic shock and multi-organ failure within 10 hours of admission. Melioidosis should be suspected in any severely ill febrile patient with an underlying predisposing condition who lives in, or has travelled from, an endemic area.

Body Mass Index is Associated with USF1 Haplotype in Korean Premenopausal Women

The upstream stimulatory factor 1 (USF1) gene has been shown to play an essential role as the cause of familial combined hyperlipidemia, and there are several association studies on the relationship between USF1 and metabolic disorders. In this study, we analyzed two single nucleotide polymorphisms in USF1 rs2073653 (306A>G) and rs2516840 (1748C>T) between the case (dyslipidemia or obesity) group and the control group in premenopausal females, postmenopausal females, and males among 275 Korean subjects. We observed a statistically significant difference in the GC haplotype between body mass index (BMI) > or =25 kg/m(2) and BMI <25 kg/m(2) groups in premenopausal females ( chi-square=4.23, p=0.04). It seems that the USF1 GC haplotype is associated with BMI in premenopausal Korean females.

Antimicrobial Resistance and Multilocus Sequence Typing of Vancomycin-Resistant Enterococcus faecium Isolated from Clinical Specimens

A total of 58 vancomycin-resistant E. faecium (VREF) was isolated from 3 hospitals located in Daegu, Korea. The VREF isolates were evaluated for the antimicrobial susceptibility pattern and resistance determinants against vancomcin, aminoglycosides, and macrolides. The multilocus sequence types (MLST) were determined to characterize the clonal diversity of the VREF isolates. The VREF isolates were highly resistance to teicoplanin, erythromycin, ciprofloxacin, gentamicin, and streptomycin, whereas quinupristin-dalfopristin and linezolid were the most susceptible drugs. All isolates carried the vanA gene. The aac6'-aph2" (n=53) and aadE (n=27) genes were detected in the high-level aminoglycoside resistant (HLAR) isolates. The aac6'-aph2" gene was located in the conjugally transferable plasmids. The ermB and ermA genes were detected in the 54 and 3 VREF isolates, respectively. The VREF isolates showed 11 different sequence types (ST). The VREF isolates belonging to ST192 was the most prevalent (n=19), but detected in one hospital, whereas the isolates belonging to ST203 (n=11) were detected in 3 hospitals. These results suggest that the VREF isolates resistant to aminoglycosides and erythromycin are originated from different clones and specific VREF clones are spread in the study hospitals.

Genetic Diversity of Stenotrophomonas maltophilia Isolated from Clinical Specimens

Stenotrophomonas maltophilia is a multi-drug resistant pathogen that has been isolated with increasing frequency from the hospitalized patients. A total of 202 S. maltophilia was isolated from three university hospitals and analysed by molecular typing for an epidemiologic investigation. All isolates were tested by antimicrobial susceptibility, random amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). The RAPD and PFGE patterns were recorded and analysed by the unweighted-pair group method with arithmetic average method. Two or more isolates were considered to be clonally related if their PFGE pattern exhibited > or =80% similarity. Trimethoprim/ sulfamethoxazole and ciprofloxacin were the most active antimicrobial agents tested. The majority of the isolates found to be genetically unrelated by PFGE. The genetically related isolates were recovered from the same patient. The result demonstrates a high genetic diversity of S. maltophilia isolates from clinical specimens. The clonal diversity of S. maltophilia from the hospitalized patients is partly due to the strains originated from the hospital environments, but not horizontal transfer between the patients

Antimicrobial Resistance and Integrons Found in Commensal Escherichia coli Isolates from Healthy Humans

The emergence and spread of antimicrobial resistance among the pathogenic and commensal Enterobacteriaceae are of great concern worldwide. We characterized the antimicrobial resistance and integrons found in commensal Escherichia coli from healthy humans in the community. Class 1 integrase (intl1) and class 2 integrase (intl2) genes were identified in 22 (13.3%) and 2 (1.2%) of 165 E. coli isolates, respectively. dfrA17-aadA5 and dfrA1-aadA2 were the most common class 1 integrons. The prevalence of each type of class 1 integron among commensal E. coli isolates during 2001~2003 was similar to that of clinical E. coli isolates from hospital-acquired infections during 1994~1999. The resistant rates of commensal E. coli isolates carrying intl1 to ampicillin, streptomycin, gentamicin, sulfamethoxazole, trimethoprim, chloramphenicol, and tetracycline were significantly higher than those of intl1-negative E. coli isolates (p<0.05). Integrons were directly associated with multidrug resistance in commensal E. coli isolates. It is hypothesized that multidrug-resistant Enterobacteriaceae from hospital-acquired infections are a potential reservoir for integrons associated with resistance genes found in commensal E. coli isolates in the community

Epidemiology of Shigellosis in Korea

Shigellosis is an acute diarrheal disease caused by bacteria of the genus Shigella. Following the occurrence of a large outbreak of shigellosis as well as sporadic cases since 1998, shigellosis has been a major health problem in Korea. There have been major changes in epidemiology during the last five decades concerning shigellosis in terms of total incidence of shigellosis, prevalence of certain serogroups, selection of specific clones, and introduction of new Shigella clones. S. dysenteriae was the most prevalent species until the early twentieth century, S. flexneri was the most prevalent until the late 1980s, and S. sonnei has been the most prevalent since 1990. Diverse serotypes of S. dysenteriae (4 serotypes), S. flexneri (8 serotypes), and S. boydii (4 serotypes) were found during the Korean War and many of these Korean endemic Shigella strains circulated in the community until the late 1970s. However, the endemic strains of S. dysenteriae, S. boydii, and S. sonnei disappeared in the late 1980s. A new clone of S. sonnei that was introduced between the late 1980s and the early 1990s was responsible for a large proportion of shigellosis in recent years. S. flexneri serotype 4a was the most frequently found during the Korean War and then the incidence of S. flexneri 2a gradually increased with time. S. flexneri isolates detected from 1991 to 1997 were all serotype 2a. However, the diverse clones of S. flexneri reemerged in Korea since 1999. It has not been determined whether the S. flexneri strains from the 2000s were the descendants of the Korean endemic strains or imported new strains, but the PFGE patterns were different between S. flexneri strains from the 1980s and 2000s. The widespread of new S. sonnei strains and the persistence of S. flexneri strains are responsible for the endemicity of shigellosis in Korea.

Prevalence of Specific Clone of Salmonella enterica Serovar Typhimurium Clones in the Swine Population of Kyungpook Province During 1998 to 2000

A total of 40 Salmolella enterica serovar Typhimurium (S. typhimurium) strains were isolated from clinical specimens of swine at 10 farms in Kyungpook province from 1998 to 2000. We investigated the clonal relationship of S. typhimurium isolates by antimicrobial susceptibility, plasmid profile, and Southern hybridization analysis with tetA, and pulsed-field gel electrophoresis (PFGE). All S. typhimurium isolates showed identical biochemical characteristics and were resistant to tetracycline, streptomycin, and sulfamehtoxazole. They were classified into 5 groups by antimicrobial resistance patterns. S. typhimurium isolates carried 3 to 5 plasmids and were classified into 5 groups by plasmid profiles. Southern hybridization showed that tetA gene was located in 21 kb of plasmid. S. typhimurium isolates from 9 different farms showed identical or similar PFGE patterns, which indicates clonal origin of the strains. All S. typhimurium isolates, except one isolate from 1998, seemed to belong to be one clone by the combination of three epidemiological typing methods. These data demonstrated that a specific clone of Salmolella enterica serovar Typhimurium was widely spread in swine farms in Kyungpook province.

Degradation of the Retinoblastoma Tumor Suppressor Protein by the Human Papillomavirus-16 E7 Variants

Human papillomavirus type 16 (HPV-16) plays an etiological role in benign and malignant epithelial tumors. A critical event in HPV transformation of human cells is the inactivation of retinoblastoma protein (pRB) by the E7 protein. The metabolic half-life of pRB is decreased in cells that express high-risk HPV E7 proteins. The present study investigated the frequency of HPV-16 E7 variants in Korean women and compared the pRB degradation activity of E7 variant proteins. Of the 40 HPV-positive specimens from a total of 91 tissue specimens, 21 HPV-16 positive specimens were studied by sequencing analysis to determine the variation of E7 gene. The most frequent E7 variant was N29S (57%). The HPV-16 E7 variant was more prevalent in invasive cervical cancer tissue specimens than in those from low grade clinical stage. The degradation of pRB in HaCaT cells by HPV-16 E7 variant proteins was investigated by western blot analysis. There was no significant difference in pRB degradation activity between the HPV-16 E7 prototype protein and E7 variant proteins. The pRB degradation activity did not differ among HPV-16 E7 variants. These results suggest that the E7-induced degradation of pRB is important in cervical tumorigenesis; however, there was no relation between the pRB degradation activity and the variations in HPV-16 E7 protein among Korean women.

Molecular Epidemiology of Nalidixic Acid Resistance in Shigella sonnei Isolates

Twenty-six nalidixic acid-resistant Shigella sonnei strains isolated from 1982 to 2001 and 56 nalidixic acid-resistant mutants induced by quinolone drugs from susceptible wild strains were analyzed by sequencing the gyrA gene. All the 22 nalidixic acid-resistant isolates from 1998 to 2001 showed identical amino acid substitution of Ser to Leu (TCG --> TTG) at codon 83 while 7 different mutation types were detected in artificially induced nalidixic acid-resistant mutants. Asp87 (GGC) type was observed most commonly among mutants induced by nalidixic acid while Ser83 (TTG) type was common among mutants induced by ciprofloxacin or norfloxacin. All the isolates collected between 1998 and 2001 showed identical or nearly identical pulsed-field gel electrophoresis pattern. These results suggest that the explosive increase of S. sonnei infection after 1998 was mainly due to the spread of restricted number of clones resistant to nalidixic acid.

Clonal Types and Antimicrobial Susceptibilitiy Patterns in Methicillin-Resistant Staphylococcus aureus Isolates from a Korean Hospital

A total of 54 non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical specimens and 3 MRSA isolates from healthy medical staffs were obtained from Kyungpook National University Hospital. They were analyzed for clonal types by multilocus sequence typing, protein A gene (spaA) typing, the staphylococcal cassette chromosome mec (SCCmec) typing, and pulsed-field gel electrophoresis (PFGE). The MRSA isolates were examined for antimicrobial susceptibility. Clinical MRSA isolates were classified into 4 clonal complexes, 4 sequence types (STs), 5 spaA types, 4 PFGE patterns, and 3 SCCmec types with variants. On the basis of ST, ST239 (n=25) and ST5 (n=24) were the most frequently encountered. MRSA isolates belonging to ST239 were genotypically homogenous, while those belonging to ST5 showed variations in spaA and SCCmec types. Of the 3 MRSA isolates from healthy medical staffs, one was genotypically identical to MRSA isolates belonging to ST5 and the other two ST239. All MRSA isolates were susceptible to vancomycin and teicoplain. Only 4% of isolates were resistant to rifampin, while 91% of isolates were resistant to ciprofloxacin. The resistance rate of MRSA isolates belonging to ST239 against trimethoprim-sulfamethoxazole (SXT) was significantly higher than that of the isolates belonging to ST5 (76% vs 0%, p<0.001). In summary, ST239 and ST5 were responsible for most MRSA infections and healthy medical staffs also carried these MRSA strains. The susceptibility of the ST239 clone against SXT, which was commonly used for oral therapy to treat MRSA infection, was significantly different from the ST5 clone.

A Comparison of ID 32 GN System with Amplified Ribosomal DNA Restriction Analysis for Identification of Acinetobacter baumannii / 대한진단검사의학회지

BACKGROUND: The glucose -acidifying genomic species 1, 2, 3 and 13 of the genus Acinetobacter are highly related genetically and may be difficult to differentiate by phenotypic identification schemes using biochemical tests. The aim of this study was to explore the brief restriction enzyme profiles of amplified ribosomal DNA restriction analysis (ARDRA) to identify medically important species of Acinetobacter. Using ARDRA analysis, we evaluated the ID 32 GN system (bioMerieux, Lyon, France) for the identification of A. baumannii (genospecies 2). METHODS: A collection of 78 A. baumannii stains initially identified by the ID 32 GN system was used to determine its accuracy by ARDRA analysis. ARDRA was performed with 10 different restriction enzymes, AluI (AGCT), CfoI (GCGC), HaeIII (GGCC), HinfI (GANTC), MboI (GATC), MspI (CCGG), NciI (CCGG), RsaI (GTAC), ScrFI (CCNGG) and TaqI (TCGA). RESULTS: The combination of restriction patterns obtained with respective enzymes AluI, CfoI and MboI allowed for the discriminatory value for the identification of medically important genospecies such as genospecies 2 (A. baumannii), 3 and 13. By comparing ARDRA results of the 78 strains previously identified by ID 32 GN system, we found the correlation rate between the two systems to be 88.5% (69/78). Nine strains were identified as Acinetobacter genospecies 13 by ARDRA. CONCLUSIONS: This result suggests that the ID 32 GN system may have difficulty in discriminating A. baumannii from genospecies 13. This revised ARDRA method gives a relatively rapid and definitive result for the identification of medically important genospecies of Acinetobacter.

Integron Carriage in Multiresistant Acinetobacter baumannii Strains

One hundred and eight Acinetobacter baumannii strains isolated from three university hospitals in Korea were investigated for the presence of integrons and its correlation with multiple antibiotic resistance. A. baumannii strains were classified into 23 different groups based on the biochemical tests, RAPD patterns, and antibiotic susceptibility. Many strains isolated from same hospital showed identical epidemiological markers: 75% of isolates from Gwangju, 53% from Seoul, and 39% from Cheonan appeared identical. Integrase genes were detected in 79 (73%) A. baumannii isolates. The intI1 and intI2 genes were detected in 73 (68%) and 10 (9%) isolates, respectively. Class 3 integrons were not detected. Integrase genes were detected in A. baumannii isolates from Gwangju and Seoul, but not in the strains from Cheonan. Based on the epidemiological grouping, most of A. baumannii strains affecting four or more patients carried integrons, while only one strain among the sporadic strains carried a class 2 integron. Integron-carrying A. baumannii strains were resistant to more antibiotics compared to the integron-negative strains. This result suggests that integron-carrying A. baumannii strains are more apt to spread nosocomially and to show multiple antibiotic resistances.

The Levels of MDM2 Protein Are Decreased by a Proteasome-Mediated Proteolysis Prior to Caspase-3-Dependent pRb and PARP Cleavages

MDM2 is a substrate of caspase-3 in p53-mediated apoptosis. In addition, MDM2 mediates its own ubiquitination in a RING finger-dependent manner. Thus, we investigated whether MDM2 is degraded through a ubiquitin-dependent proteasome pathway in the absence of p53. When HL-60 cells, p53 null, were treated with etoposide, MDM2 was markedly decreased prior to caspase-3-dependent retinoblastoma tumor suppressor protein (pRb) and poly (ADP- ribose) polymerase (PARP) cleavages. Moreover, down-regulation of MDM2 level was not coupled with its mRNA down-regulation. However, the level of MDM2 was partially restored by proteasome inhibitors such as LLnL and lactacystin, even in the presence of etoposide. Our results suggest that, in the p53 null status, MDM2 protein level is decreased by proteasome-mediated proteolysis prior to caspase-3-dependent PARP and pRb cleavages.