beta-Lapachone suppresses radiation-induced activation of nuclear factor-kappaB

Anticancer effects of beta-lapachone (beta-lap) are due to generation of ROS and metabolic catastrophes as a result of NAD(P)H:quinone oxidoreductase (NQO1)-mediated futile cycling between the oxidized and reduced forms of beta-lap. It has been shown that NQO1 is also essential for the TNF-induced activation of NF-kappaB and that beta-lap suppresses the TNF-induced NF-kappaB activation. We investigated whether or not NQO1 is involved and beta-lap suppresses the radiation-induced NF-kappaB activation using A549 human lung cancer cells and NQO1-knock down A549 cells (shNQO1 A549 cells). Irradiation with 4 Gy markedly increased the DNA binding activity of NF-kappaB in A549 cells, but not in the shNQO1 A549 cells, thus demonstrating that NQO1 plays a pivotal role in irradiation-induced NF-kappaB activation. Treatment with 10 micrometer beta-lap for 4 h almost completely abrogated the radiation-induced increase in NF-kappaB activation and the transcription of NF-kappaB target genes such as bcl2, gadd45beta and cyclinD1. Moreover, beta-lap markedly suppressed the activation of IkappaB kinase gamma (IKKgamma) and the subsequent phosphorylation of IkappaBalpha, thereby inhibiting NF-kappaB activation. It is concluded that beta-lap suppresses the radiation-induced activation of NF-kappaB by interrupting the involvement of NQO1 in the activation of NF-kappaB, thereby inhibiting the transcription of survival signals. The radiosensitization caused by beta-lap may, in part, be attributed to beta-lap-induced suppression of NF-kappaB activation.

Association of the GSTP1 and NQO1 Polymorphisms and Head and Neck Squamous Cell Carcinoma Risk

The GSTP1 and NQO1 have been reported to be associated with an increased risk for smoking related head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to determine the effect of these metabolic gene polymorphisms on the risk of HNSCC. The study population included 294 histologically confirmed HNSCC cases and 333 controls without cancer. Genotyping analysis of the GSTP1 Ile105Val and NQO1 Trp139Arg genes was performed by polymerase chain reaction-based techniques on DNA prepared from peripheral blood. The Mantel-Haenszel chi-square test was used for statistical analysis. The allele frequencies of the GSTP1 and NQO1 polymorphisms were not statistically significant between cases and controls. In analyzing the association between smoking amounts and genetic polymorphisms, GSTP1 and NQO1 polymorphisms were associated with cigarette smoking amounts in cases. G allele containing genotypes in GSTP1 and T allele containing genotypes in NQO1 were associated with a tobacco dose-dependent increase in risk of HNSCC and these genotype distributions were statistically significant (p<0.05). We found that the GSTP1 105Val allele and NQO1 139Arg allele were associated with tobacco dose-dependent increase in risk of HNSCC. GSTP1 and NQO1 genotype polymorphisms may play an important role in the development of smoking related HNSCC.

Synergistic Effect of Ionizing Radiation and beta-lapachone against RKO Human Colon Adenocarcinoma Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: To reveal the interaction between beta-Lapachone (beta-lap) and ionizing radiation in causing cell death in RKO human colon adenocarcinoma cells, and to elucidate the potential usefulness of combined beta-lap treatment and radiotherapy for cancer treatment. MATERIALS AND METHODS: The cytotoxicities of various treatments were determined in vitro using clonogenic and apoptotic cell death. The changes in cell cycle distribution were studied using flow cytometry and an in vitro kinase assay. The tumor growth was studied using RKO tumors grown s.c. in the hind leg BALB/c- nuslc nude mice. RESULTS: beta-lap caused clonogenic cell death and rapid apoptosis in RKO cells in vitro, in a dose dependent manner. The repair of sublethal radiation damage was almost completely inhibited when cells were maintained in beta-lap during the interval between the two-dose irradiation. Flow cytometry study demonstrated that beta-lap induced apoptosis, independent of the cell cycle phase, and completely prohibited the induction of radiation- induced G2 arrest in irradiated cells. The prohibition of radiation-induced G2 arrest is unclear, but may be related to the profound suppression of the p53, p21 and cyclin B1-Cdc2 kinase activities observed in cells treated with beta-lap. The combination of beta-lap and radiation markedly enhanced the radiation-induced growth suppression of tumors. CONCLUSION: beta-lap is cytotoxic against RKO cells, both in vitro and in vivo, and also sensitized cells to ionizing radiation by inhibiting sublethal radiation damage repair. beta-lap is potentially useful as a potent anti-cancer chemotherapy drug and potent radiosensitizer against caner cells.

Polymorphisms of DNA Repair Gene XRCC1 and Radiation Sensitivity

PURPOSE: The goal of this study was to find lung cancer-related single nucleotide polymorphisms (SNP) and define their association with clinical results. Material and Methods: One hundred and thirty-six non-small cell lung cancer patients, who received radiotherapy at the Asan Medical Center, were recruited between August 2002 and September 2003. Demographic and clinical informations were obtained from a self-administered questionnaire and from the subject's medical records, respectively. Blood samples were collected from all study subjects at the time of enrollment. Genomic DNA was extracted from peripheral blood lymphocytes using a QIAamp DNA Blood Mini Kit. TaqMan assay, denaturing HPLC and single base pair primer extension assay using SNaPshot kits were employed as the SNP screening techniques. The candidate SNP for screening was XRCC1-R399Q. RESULTS: Patients carrying the 399Gln variant allele had a significantly longer progression-free survival than those with the 399Arg homozygote in tumor stages I-IIIa (p=0.005). In the Cox-proportional hazards model, the XRCC1 codon 399 polymorphism was a statistically significant predictor for progression-free survival in tumor stages I-IIIa (p=0.03). CONCLUSION: The use of molecular predictors of the progression-free survival in non-small cell lung cancer patients, particularly at stages I-IIIa, may provide important criteria for prognosis of the patients undergoing radiotherapy. However, there is still a need for further study to establish the role of these polymorphisms as useful predictors

Clinical Results of Stereotactic Body Frame based Fractionated Radiosurgery for Primary or Metastatic Thoracic Tumors

PURPOSE: The aim of the study was to evaluate the treatment outcomes of stereotactic radiosurgery (SRS) using a stereotactic body frame for primary or metastatic thoracic tumors. Methods and Materials: Between January 1998 and December 2003, 101 lesions from 91 patients with primary or metastatic thoracic tumors were treated. The eligible patients included 38 with primary lung cancers and 53 with metastatic tumors from the lung, liver, gastrointestinal and other organs. All patients were immobilized using a stereotactic body frame and permitted to breathe shallowly. The respiratory movement was restricted by a diaphragm controller when the tumor movement was greater than 5 mm. Recently, for further restriction of tumor movement, an active breathing control (ABC) apparatus was used in some trained patients whose tumors located in lower lobe. Three to eight coplanar or non-coplanar photon beams were used to adequately cover the planning target volume. A dose of 10~12 Gy per fraction was given three to four times over consecutive days, to a total dose of 30~48 Gy (median 40 Gy). Local control was assessed as complete or partial responses and by a stable disease, as measured by serial chest CT scans at 1 month, and then every 3-months, and/or 18FDG-PET scans 1 month after treatment. The median follow-up period was 14 months, ranging from 4 to 56 months. RESULTS: The overall response rate was 82%, with twenty (22%) complete and 55 (60%) partial responses. The rate of crude local control in all patients was 86% and the one- and two-year local progression free survival rates were 90 and 81%, respectively. The patients who received 48 Gy showed better local progression free survival than those that received 40 Gy or less (one-year; 100% vs. 86.7%), but this was not statistically significant. Of the 21 patients with primary lung cancer, local progression was observed in 3, at 12, 21 and 26 months after treatment, and the one- and two- year local progression free survival rates were 93 and 81%, respectively. The set-up error, as checked by CT-simulation and portal films, for every treatment was within 5 mm in all directions (X, Y and Z axis). No pulmonary complications greater than RTOG toxicity criteria grade 2 were observed. CONCLUSION: From our experience of the stereotactic body frame based radiosurgery it appears a safe and promising treatment modality for the local management of primary or metastatic lung tumors. The optimal total dose, fractionation schedule and treatment volume should be modified after a longer follow-up of these results. Further study related to the optimal evaluation tools is also necessary to differentiate local tumor progression from radiation-induced pulmonary injury

Association between Expression of NAD(P)H: Quinone Oxidoreductase 1 (NQO1) and Synergistic Effect of Ionizing Radiation and beta-lapachone on Human Lung Cancer Cell Line (A549)

PURPOSE: beta-lapachone ( beta-lap) has cytotoxic effect in a number of human cancer cell lines and through a unuique apoptotic pathway that is dependent upon NQO1, a two-electron reductase. beta-lap was an effective agent alone, and in conbination with radiation or taxol with low-level host toxicity. We investigated the cytotoxicity of beta-lap against A459 human lung cancer cells as well as the combined effect of beta-lap and ionizing radiation. MATERIALS AND METHODS: An incubation of A459, HN3 and HN9 cells with 5 M of beta-lap for 4 h killed almost 90% of the clonogenic cells. Raidation and beta-lap acted synergistically in including clonogenic cell death and apoptosis in A549 cells when beta-lap treatment was applied after but not before the radiation exposure of the cells. Interestingly, a 4 h treatment with 5 M of beta-lap starting 5 h after irradiation was as effective as that stating immediately after irradiation. The mechanisms of beta-lap induced cell killing is supposed that beta-lap is activated by NAD(P)H: quinone-oxidoreductase (NQO1) in the cells followed by an elevation of cytosolic Ca2+ level and activation of proteases leading to apoptosis. RESULTS: We observed that beta-lap killed human lung and head neck cancer cells by an apoptotic response significantly enhanced by NAD(P)H: quinone oxidoreductase (NQO1) enzymatic activity after 4 Gy irradiation. CONCLUSION: Taken together, we may conclude that the synergistic interaction of radiation and beta-lap in killing cancer cells is not due to radiosensitization by beta-lap but to enhancement of beta-lap cytotoxocity by radiation through upregulation of NQO1

Molecular Aspects of Radiotherapy

When tumor cells are exposed to ionizing radiation, various and complicated molecular biological changes take place leading to cell death, mutation, and recovery from sublethal damage. It has been known that DNA is the major critical target of radiation leading to cell death. The radiation-induced DNA damage activates ATM/ATR which then lead to activation and phosphorylation of downstream molecular signals, such as p53. Phosphorylation of p53 leads to inhibition of cell cycle progression, cell death through apoptosis and repair of damaged DNA. Recent evidence clearly demonstrated that p53 is directly involved in activation of cell cycle checkpoints resulting in G1 arrest and G2 arrest. During these arrests, the damaged DNA are repaired. However, when the radiation-induced DNA damage is excessive, cells undergo apoptotic cell death. Here again, p53 is involved in activation of pro-apoptotic signals such as Bax and caspases and inactivation of anti-apoptotic signals such as Bcl-2. Proper activation or intervention of these molecular signals may enable us to enhance the radiation damage in cancer cells and improve the efficacy of radiotherapy of malignant cancer.

Preliminary Results of Stereotactic Body Frame Based Fractionated Radiosurgery on Consecutive Days for Primary Lung Cancer or Metastatic Lung Tumors

To evaluate the feasibility and treatment outcomes of stereotactic radiosurgery (SRS) using a stereotactic body frame (Precision TherapyTM), we prospectively reviewed 34 tumors of the 28 patients with primary or metastatic intrathoracic lung tumors. Eligible patients included were 9 with primary lung cancer and 19 with metastatic tumors from the lung, liver and many other organs. A single dose of 10 Gy to the clinical target volume (CTV) was delivered to a total dose of 30~40 Gy with 3~4 fractions. Four to 8 coplanar or non-coplanar static fields were generated to adequately cover the planning target volume (PTV) as well as to exclude the critical structures as much as possible. More than 90% of the PTV was delivered the prescribed dose in the majority of cases (average; 96%, range; 74%~100%). The mean PTV was 41.4 cc ranging from 4.4 to 230 cc. Set-up error was within 5 mm in all directions (X, Y, Z axis). The response was evaluated by using a chest CT and or 18FDG-PET scans after SRS treatment, 11 patients (39%) showed complete response, 12 (43%) partial response (decrease of more than 50% of the tumor volume), and 4 patients showed minimally decreased tumor volume or stable disease, but 1 patient showed progressive disease. With a median follow-up period of 18 months, a median local disease progression free interval was 18 months ranging from 7 to 35 months. Although all patients developed grade 1 radiation pneumonitis within 3 months, none had symptomatic or serious late complications after completing SRS treatment. Given these observations, it is concluded that the stereotactic body frame based SRS is a safe and effective treatment modality for the local management of primary or metastatic lung tumors. However, the optimum total dose and fractionation schedule used should be determined after the longer follow-up of these results.

Subpopulation of Rabbit Articular Chondrocytes Separated by Percoll Density Gradient / 대한정형외과연구학회지

Articular chondrocytes have been known to have heterogeneity in articular cartilage. Four layers are generally recognized from the articular surface to the subchondral bone. We have used Percoll density gradients to separate chondrocytes from articular cartilage into distinct subpopulations. Non-fibrillated articular cartilage was obtained from rabbit knee. The cells were carefully layered on the top of the preformed gradient and spun. After centrifugation, we obtained four fractions: Fraction A referred boundary between 0% and 10%, fraction B from between 10% and 20%, fraction C from between 20% and 30%, and fraction D from between 40% and 50%. In the A fraction, cells are relatively larger and round in shape, while their nuclei are relatively smaller. In the cytoplasm many lipid droplets were found and rough endoplasmic reticulum were disrupted. In the D fraction, chondrocyte is small, with large nucleus which surrounded by well-developed rough endoplasmic reticulum. The type II collagen proteins were expressed strongly and more proteoglycans synthesized in fractions A and B. And chondrocytes from the fraction D divided more slowly than those from the fractions A, B, and C. We have succeeded in separating chondrocytes from articular cartilage into distinct subpopulations by Percoll density gradients, as well as characterized growth rate, histological appearances and phenotypic expression. This study is the first report about the Percoll density gradients to separate articular chondrocytes.

Radiation-induced Apoptosis is Differentially Modulated by PTK Inhibitors K562 Cells / 대한방사선종양학회지

PURPOSE: The effect of PTK inhibitors (herbimycin A and genistein) on the induction of radiation-induce d apoptosis in Ph-positive K562 leukemia cell line was investigated. MATERIALS AND METHODS: K562 cells in exponential growth phase were irradiated with a linear accelerator at room temperature. For 6 MV X-ray irradiation and drug treatment, cultures were initiated at 2x10' cells/mL. The cells were irradiated with 10 Gy. Stock solutions of herbimycin A and genistein were prepared in dimethyl sulphoxide (DMSO). After incubation at 37C for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and TUNEL assay. The progression of cells throughth the cel l cycle after irradiation and drug treatment was also determined with flow cytometry. Western blot analysis was used to monitor bcl-2, bcl-X and bax protein levels. RESULTS: Treatment with 10 Gy X-irradiation did not result in the induction of apoptosis. The HMA alone (500 nM) also failed to induce apoptosis. By contrast, incubation of K562 cells with HMA after irradiation resulted in a substantial induction of nuclear condensation and fragmentation by agarose gel electro-phoresis and TUNEL assay. Genistein failed to enhance the ability of X-irradiation to induce DN A fragmentation. Enhancement of apoptosis by H MA was not attributable to downregulation of the bcl-2 or bcl-X anti-apoptotic proteins. When the cells were irradiated and maintained with HMA, the percentage cf cells in G2/M phase decreased to 30-40% at 48 h. On the other hand, cells exposed to 10 Gy X-irradiation alone or maintained with genistein did not show marked cell cycle redistribution. CONCLUSION: We have shown that nanomolar concentrations of the PTK inhibitor HMA synergize with X-irradiation in inducing the apoptosis in Ph (+) K562 leukemia cell line. While, genistein, a PTK inhibitor which is not selective for p2 10""'' failed to enhance the radiation induced apoptosis in K562 cells. It is unlikely that the ability of HMA to enhance apoptosis in K562 cells is attributable to bcl-2 family. It is plausible that the relationship between cell cycle delays and cell death is essential for drug development based on molecular targeting designed to modify radiation-induced apoptosis.

The Expression of Oncogenes on the Radiation-induced Apoptosis in SCK Mammary Adenocarcinoma Cell Line / 대한방사선종양학회지

PURPOSE:The expression of p53, p21/WAF/CIP, Bcl-2, and Bax underlying the radiation-induced apoptosis in different pH environments using SCK mammary adenocarcinoma cell line was investigated. MATERIALS AND METHODS: Mammary adenocarcinoma cells of A/J mice (SCK cells) in exponential growth phase were irradiated with a linear accelerator at room temperature. The cells were irradiated with 12 Gy and one hour later, the media was replaced with fresh media at a different pHs. After incubation at 37 degrees C for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and flow cytometry. The progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. Western blot analysis was used to monitor p53, p21/WAF/CIP, Bcl-2, and Bax protein levels. RESULTS:The induction of apoptosis by irradiation in pH 6.6 medium was markedly less than that in pH 7.5 medium. The radiation-induced G2/M arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. Considerable amounts of p53 and p21 proteins already existed at pH 7.5 and increased the level of p53 and p21 significantly after 12 Gy X-irradiation. An incubation at pH 6.6 after 12 Gy X-irradiation did not change the level of p53 and p21 protein levels significantly. Bcl-2 proteins were not significantly affected by radiation and showed no correlation with cell susceptibility to radiation-induced apoptosis in different pHs. An exposure to 12 Gy of X-rays increased the level of Bax protein at pH 7.5 but at pH 6.6, it was slight. CONCLUSION: The molecular mechanism underlying radiation-induced apoptosis in different pH environments using SCK mammary adenocarcinoma cell line was dependent of the expression p53 and p21/WAF/CIP proteins. We may propose following hypothesis that an acidic stress augments the radiation-induced G2/M arrest, which inhibiting the irradiated cells undergo post-mitotic apoptosis. The effects of environmental acidity on anti-apoptotic and pro-apoptotic function of Bcl-2 family was unclear in SCK mammary adenocarcinoma cell line.

Apoptosis of Murine Macrophage-like Cells Infected with Orientia tsutsugamushi

A large number of bacterial pathogens have been identified as mediators of apoptosis in vitro and the induction of apoptosis might be an important step in the pathogenesis of these bacteria. In this study, we analyzed the interactions of Orientia tsutsugamuchi with J774 murine macrophage-like cells. The J774 cells were infected with Boryong strain of O. tsutsugamushi and the DNA was analyzed with agarose gel electrophoresis. We observed the typical laddering pattern of DNA fragmentation indicative of apoptosis in infected cells but not cells infected with heat-killed O. tsutsugamushi. We performed terminal deoxynucleotidyl transferase (TdT) assay to label the 3'-hydroxy ends of DNA breaks and observed intense brown staining of nuclei of infected macrophages. With Hoechst 33258 for staining nucleus, strong chromatin condensation was observed only in infected J774 cells. We also examined the cytokine secretion pattern of J774 cells during the rickettsial infection. The large amount of TNF-alpha and IL-10 were secreted after 24 hrs of infection, but the secretion of IL-1beta was increased in small amount. These results showed that O. tsutsugamushi induce apoptosis in murine macrophage-like cells by different mechanism from that of shigella which cause secretion of large amount of IL-1beta.

The Cell Cycle Dependence and Radiation-induced Apoptosis in SCK Mammary Adenocarcinoma Cell Line / 대한방사선종양학회지

PURPOSE: The relationship between environmental pH on the radiation induced-apoptosis in SCK mammary adenocarcinoma cells and cell cycle dependence was investigated. MATERIAL AND METHODS: Mammary adenocarcinoma cells of A/J mice (SCK cells) in exponential growth phase were irradiated with a 137Cs irradiator at room temperature. The cells were irradiated 1 hour after the media was replaced with fresh media at a different pHs. After incubation at 37degrees C for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and flow cytometry. The progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. RESULTS: The induction of apoptosis by irradiation in pH 6.6 medium was markedly less than that in pH 7.5 medium. When the cells were irradiated and maintained in pH 7.5 medium, the percentage of cells in G2/M phase rapidly increased to about 70% at 12 h after an exposure to 12Gy and returned to control level by 36 h. The percentage of cells in G1 phase decreased as the percentage of cells in G2/M increased. On the other hand, in pH 6.6 medium the percentage of cells in G2/M phases gradually increased to about 45% at 24 h after 12Gy irradiation and then slowly recessed and consequently, as much as 30-35% of the cells were still in the G2/M phase 48 h after irradiation. The percentage of cells in G1 phase then increased as the G2/M arrest began to recede. The radiation-induced G2/M arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. CONCLUSION: Radiation-induced apoptosis in SCK tumor cells are reversely suppressed in an acidic environment. Radiation-induced G2/M arrest is prolonged in an acidic environment indicating that the suppression of radiation- induced apoptosis and prolongation of radiation-induced G2/M arrest in an acidic environment are related.

The Cell Cycle Dependence and Radiation-induced Apoptosis in SCK Mammary Adenocarcinoma Cell Line / 대한방사선종양학회지

PURPOSE: The relationship between environmental pH on the radiation induced-apoptosis in SCK mammary adenocarcinoma cells and cell cycle dependence was investigated. MATERIAL AND METHODS: Mammary adenocarcinoma cells of A/J mice (SCK cells) in exponential growth phase were irradiated with a 137Cs irradiator at room temperature. The cells were irradiated 1 hour after the media was replaced with fresh media at a different pHs. After incubation at 37degrees C for 0-48 h, the extent of apoptosis was determined using agarose gel electrophoresis and flow cytometry. The progression of cells through the cell cycle after irradiation in different pHs was also determined with flow cytometry. RESULTS: The induction of apoptosis by irradiation in pH 6.6 medium was markedly less than that in pH 7.5 medium. When the cells were irradiated and maintained in pH 7.5 medium, the percentage of cells in G2/M phase rapidly increased to about 70% at 12 h after an exposure to 12Gy and returned to control level by 36 h. The percentage of cells in G1 phase decreased as the percentage of cells in G2/M increased. On the other hand, in pH 6.6 medium the percentage of cells in G2/M phases gradually increased to about 45% at 24 h after 12Gy irradiation and then slowly recessed and consequently, as much as 30-35% of the cells were still in the G2/M phase 48 h after irradiation. The percentage of cells in G1 phase then increased as the G2/M arrest began to recede. The radiation-induced G2/M arrest in pH 6.6 medium lasted markedly longer than that in pH 7.5 medium. CONCLUSION: Radiation-induced apoptosis in SCK tumor cells are reversely suppressed in an acidic environment. Radiation-induced G2/M arrest is prolonged in an acidic environment indicating that the suppression of radiation- induced apoptosis and prolongation of radiation-induced G2/M arrest in an acidic environment are related.

Dedifferentiation of Human Chondrocyte in Monolayer Culture and its Reversibility in Pellet Culture / 대한정형외과연구학회지

Articular cartilage is a unique tissue devoid of blood and nerve tissue and so its regeneration is very limited. Recently a clinical trial on transplantation using autologous chondrocyte with periosteal flap has drawn a great deal of attention. Chondrocytes cultured in a plastic flask in monolayer can rapidly dedifferentiate appearing fibroblastic, and exhibit a change in matrix gene expression characterized by a decrease in type II collagen synthesis. It is uncertain whether phenotypic change of dedifferentiated chondrocytes in vitro can be reversible to their original status alter long term culture. It is important to verify tile maintenance of the phenotype and determine the optimum period for culturing chondrocytes to be used in autologous chondrocyte transplantation. This study will be set up to confirm the reversibility of once-dedifferentiated chondrocytes with matrix-producing capability. The phenotype of cultured human chondrocyte is analysed by Northern blot and Western blot analysis for collagen type I and II. Chondrocytes appeared fibroblast right after adhering to the flask buttom at first week of culture. The proliferating rates of chondrocyte in a monolayer culture were maximum at 3rd and 4th week of culture. And thereafter, proliferation rate flowed down or stops as confluence rose. On Northern and Western blot analysis, collagen type II was well expressed by 3th to 4th week culture, thereafter progressively decreased its density with time. On the other hand, collagen type I m-RNA has not expressed until 3rd week of the culture, showing progressive increment of density thereafter. On Northern blot analysis in pellet culture, type II collagen m-RNA is apparantly reexpressed. This study indicates chat in the monolayor culture, the chondrocytic phenotype was lost with regards to morphology and mRNA expression and cartilage specific protein. However, these cells seemed to haute the potential to redifferentiate to well-differentiated chondrocytes in densely packed culture, such as pellet.

Effects of intracellular pH on apoptosis in HL-60 human leukemia cells

The nature of the endonucleases responsible for DNA fragmentation in apoptosis has not yet been clearly defined. The intracellular acidity has been known to greatly affect apoptosis probably by affecting the activity of the endonucleases. In this study, the implication of pH in the apoptosis was investigated through the use of human HL-60 leukemia cells. The cells were incubated in media with different pH ranging from 3.5 to 7.5 for 4 hrs and the mode of cell death was investigated. The trypan blue exclusion assay showed that close to 25% and 90% of the cells were dead when incubated in pH 6.4 and pH 5.0 media, respectively. The agarose gel electrophoresis of DNA demonstrated that significant DNA fragmentation occurred in the HL-60 cells incubated in the pH 6.2-6.4 media for 4 hr indicating cell death by apoptosis. The electron microscopy study also demonstrated that many of the cells incubated in the pH 6.4 medium were in the process of apoptosis while the cells maintained in the pH 5.0 medium were dying by necrosis. The intracellular pH (pHi) of HL-60 cells was 6.6-6.9 when the extracellular pH (pHe) was 6.2-6.4. These results demonstrated that DNase I which has a maximal endonuclease activity near pH 7.0 may be responsible for the apoptosis accompanied by DNA fragmentation in HL-60 cells in the pH 6.4 medium. This observation is at variance with the previous reports that DNase II mediate the DNA fragmentation in apoptosis. The cell death at extremely low pH (pH 5.0) appeared to be due mainly to necrosis.