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The Journal of Nutrition Vol. 129 No. 1 January 1999,
pp. 25-31
, and
* Department of Nutrition and Food Sciences, Fu-Jen University, Hsin-Chuang, Taiwan, Graduate Institute of Basic Medicine, School of Medical Technology, Chang Gung University, Taoyuan, Taiwan and ** Department of Medical Research, Yuan's General Hospital, Kaohsiung, Taiwan, R.O.C.
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The human hepatoma HepG2 cell line was chosen as a representative of solid tissue-derived cell systems in which folate metabolism and apoptosis induction have not been thoroughly investigated. HepG2 cells were cultivated in the control or folate-deficient media (control media lacking of folate, glycine, thymidine and hypoxanthine) for 4 wk. This resulted in a decrease in intracellular folate levels to 32% of the control within 1 wk, which was followed by growth arrest and greater cell death rates. These disturbances of folate deficiency coincided with apoptotic induction, as characteristically shown by nucleosomal DNA fragmentation of 180-200 base pair multimers, nuclear chromatin condensation and positive terminal transferase-mediated dUTP nick end labeling assay. Apoptosis coincided with an accumulation of cells in S-phase, a subsequent G2/M phase block and a significant increase in mean protein content as evaluated by flow cytometric analyses employing a double-staining method. The growth and cell cycle arrest under folate-deficient conditions was independent of a change of p53 expression as measured by an enzyme-linked immunosorbent assay. Supplementation of 2 µmol/L folate normalized cell cycles and diminished DNA fragmentation. Taken together, these data indicate that HepG2 cells cultivated in folate-deficient medium have a low folate concentration, decreased growth and viability, and increased apoptotic propensity. This occurrence of apoptosis was associated with a cell cycle-specific mechanism and independent of p53-mediated pathway.
KEY WORDS: folate deficiency · HepG2 cells · apoptosis · cell cycle arrest · DNA fragmentation
Folate coenzymes are critical for de novo synthesis of purines and thymidine, and for interconversion of amino acids. Folate deficiency inhibits cellular proliferation, disturbs cell cycling (Borman and Branda 1989 One mode of cell death, apoptosis, is the universal mechanism by which undesirable cells are cleared to maintain homeostasis of cell turnover. Apoptotic cells exhibit characteristics of cytoskeletal disruption, cell shrinkage, membrane blebbing and DNA fragmentation to 180-200 base pair multimers (reviewed in Compton 1992 The relationship between folate deficiency and the occurrence of cell death through apoptosis has not been well characterized. Cellular consequences of apoptosis due to folate deficiency also are not clearly defined. To fill in these gaps, this study was conducted to explore the possible role of folate deficiency in the mechanism(s) of cell death, using the human hepatoma HepG2 cell line, a representative of solid tissue-derived cell systems, as an experimental model.
Materials.
Folate (pteroylmonoglutamic acid)5, amino acids, nucleosides, nucleotides and other chemical compounds were purchased from Sigma Chemical (St. Louis, MO). Minimal essential medium/alpha modified ( Cell culture.
The human hepatoma HepG2 cell line was obtained from the National Development Center of Biotechnology (Taipei, Taiwan). HepG2 cells were maintained as monolayer culture in complete medium at 37°C in a humidified 5% CO2 incubator with medium changed every 2 d. Complete media contained Cell proliferation and cell death.
Confluent HepG2 cells growing in complete media were replated into 60-mm culture dishes in the presence of control or folate-deficient medium. Regular subculture of each treatment was performed at the end of each week. For growth studies, each week of folate-deficient or control culture was subcultured into various sets of plates (seeding cell density: 5-6 × 104 cells/35 mm dish). On every other day, cells in triplicate plates were trypsinized. Viable cells were identified by trypan blue exclusion and cell numbers were counted by hemocytometer. Growth curves were plotted as the logarithm of total cell numbers versus culture time. Population doubling times were calculated from the slope of the linear portion of each growth curve and defined as log2/slope. Relative maximum growth rates were calculated as the ratio of doubling time in control medium/doubling time in folate-deficient media. Cell death rates were expressed as the percentage of nonviable cells stained by trypan blue over total cell numbers in the triplicate plates.
Intracellular folate.
Intracellular folate levels were determined by the method of Horne and Patterson (1988) Analysis of cell cycles and proteins.
Double staining method for DNA and protein was modified from the procedure of Borman and Branda (1989) Electron microscopy examination.
The ultrastructural characteristics of folate-deficient or control cells were visualized by transmission electron microscopy. Cell cultures were prefixed with 2.5% glutaraldehyde and 2% paraformaldehyde at 4°C for 1-2 h. The cells were then postfixed with 1% osmium tetroxide, dehydrated in an ethanol series and embedded in spurr epon (Spurr 1969 Agarose electrophoresis of DNA fragmentation.
DNA fragmentation was assayed by the modification of methods as previously described by James et al. (1994) Terminal transferase-mediated dUTP nick end labeling (TUNEL) assay.
TUNEL assay was performed as described in Gold et al. (1993) ELISA assay for p53 protein.
In parallel with analysis of cell cycle progression and apoptosis, replicate cultures were processed for determination of p53 protein levels by ELISA. Protein lysates were extracted from 2 × 106 cells cultured in control or folate-deficient media using lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L sodium chloride, 10 mmol/L EDTA, 1% nonylphenyl-polyethyleneglycol, 100 mmol/L phenyl methyl sulphonyl fluoride, 100 mmol/L sodium orthovanadate). Sandwich enzyme immunoassays (pantropic p53 quantitative ELISA, Oncogene Science, Cambridge, MA) were used according to the manufacturer's instructions.
Statistics.
Data were analyzed to determine statistical significance of differences between the control and folate-deficient groups by Student's paired t test. A P value of <0.05 is considered significant.
Kinetics of intracellular folate levels, cellular proliferation and death rates under folate deficiency.
By the L. casei assay, folate levels in control cells (252 ± 37 pmol/106 cells) were not different from those cultured in complete medium (241 ± 14 pmol/106 cells). HepG2 cells cultured in folate-deficient media for 4 wk had depleted intracellular folate levels. After 1 wk of folate-deficient culture, HepG2 cells had intracellular folate levels that were 32% of control (Fig. 1A). This deficit of intracellular folate persisted for the remainder of culture times. Folate-deficient HepG2 cells exhibited a folate-dependent loss of proliferation capacity. The relative growth rates of folate-deficient cells were reduced to 74 ± 5% of control at the first week and became cytostatic thereafter (16 ± 9% by wk 2 and 4 ± 3% by wk 3) (Fig. 1A). Accompanying growth arrest, greater cell death rates in the folate-deficient population were observed (Fig. 1B).
Apoptosis induced by folate deficiency.
Culture of HepG2 cells under folate deficienct conditions for at least 2 wk caused DNA damage. Figure 2 shows the DNA fragmentation pattern visible in the electrophoretogram of cells cultured in complete, control or folate-deficient media. DNA extracted from cells cultured in complete (lane 0) or control media from 1 to 4 wk (lanes 1-4) was shown to be intact. DNA from folate-deficient cells was fragmented into 200 base pairs of nucleosomal multimers typical of oligonucleosomal ladders. DNA ladders were evident after wk 2 and 3 of folate-deficient culture (Deficient: lanes 2 and 3) but will not present by wk 4 of deficiency. Instead, large DNA fragments were visible (lane 4), suggesting a second phase of DNA damage in the surviving cells. The apoptosis of folate-deficient cells was further confirmed by transmission electron microscopy which revealed nuclear chromatin condensation in folate-deficient cells (Fig. 3). Taken together, these data indicated that folate-deficient cells underwent apoptosis.
Perturbation of DNA distribution and cell cycles.
DNA distribution histograms and analysis of cell cycles are shown in Figure 4. As intracellular folate dropped and growth inhibition started in folate-deficient cells, a pronounced change in cell cycling of the HepG2 cells was observed (Fig. 4A). At wk 1 of folate deficiency, HepG2 cells had a greater percentage of cells in S phase compared to that of the control (Fig. 4B, middle). Then the S phase-arrest effect was more pronounced as folate deficiency was prolonged to wk 2 and 3. The proportion of S phase cells in folate-deficient cultures was 100% greater than those in control cultures (Fig. 4B, middle). The increase in the percent of folate-deficient cells in S phase was initially accompanied with decreased G0/G1 phase cells (Fig. 4B, top) and subsequently with G2/M phase cell accumulation (Fig. 4B, bottom). During this folate-deficient period, cells arrested in S-phase and G2/M phase predominated in the folate-deficient HepG2 cultures (Fig. 4B). The data indicated that folate deficiency caused S phase cell accumulation and G2/M phase block.
Perturbation of protein contents.
The mean green fluorescence intensity was proportional to relative mean protein content in cells analyzed by flow cytometry. At 2 wk of folate deficiency, the green fluorescence intensity in folate-deficient cultures was shifted to higher relative mean protein content compared to control cultures and continued to increase during wk 3 and wk 4 of folate deficiency (Fig. 5A). Absolute protein levels in folate-deficient cells were significantly greater than those in control cells at wk 2-4 (Fig. 5B). The data suggested that protein synthesis in arrested HepG2 cells was not inhibited.
p53 levels during folate-deficient derived apoptosis.
The p53 levels in folate-deficient cells were not significantly different from those in control cells during wk 1-4 of culture (Fig. 6), indicating that apoptosis of folate-deficient HepG2 cells was independent of increased p53 expression.
Effects of folate supplementation on apoptosis.
The positive signals in the TUNEL analysis of 3 wk folate-deficient cultures provided additional evidence of apoptosis (Fig. 7A). The apoptotic cultures could be rescued at wk 3 of deficiency by administration of 2 µmol/L folate for 3 wk, wherein DNA fragments gradually disappeared (Fig. 7A). In parallel with the disappearance of DNA fragments, the cell cycles of the S-arrested cells were normalized after folate supplementation for 23 d (Fig. 7B).
By using gel electrophoresis, the TUNEL assay and electron microscopy to detect DNA fragmentation and chromatin condensation, we demonstrated that folate deficiency induced apoptosis in human hepatoma HepG2 cells. The apoptosis was preceded by a drop of intracellular folate content and accompanied by a cell cycle block as well as increased cell death. Folate supplementation could rescue apoptotic cells from further damage and normalize cellular growth (Ho and Huang 1997
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
), causes genetic damage (Branda and Blickensderfer 1993, Libbus et al. 1990) and eventually results in cell death. The underlying cell death mechanisms associated with these observed phenomena remain elusive.
). Following the delivery of a variety of stimuli to the cells, apoptosis involves the regulation of gene transcription or molecular activation to execute multiple cellular self-destruction processes. For example, certain types of DNA damage trigger apoptosis through p53-dependent signal transduction pathway (reviewed in Bellamy 1997). P53, a tumor suppressor protein, functions to regulate the expression of genes associated with growth arrest or programmed cell death (Yonish-Rouach et al. 1993, Yonish-Rouach 1996). Deregulation of p53-mediated apoptotic processes may be involved in oncogenesis (reviewed in Wang and Harris 1997), and apoptosis may be associated with many human diseases, including cancer (Thompson 1995).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
MEM) without ribosides, ribotides, deoxyribosides, deoxyribotides, glycine, serine and folate was specially ordered and formulated by JRH (Lenex, KS). Fetal bovine serum (FBS) was from HyClone Laboratories (Logan, UT). Penicillin, streptomycin, fungizone, trypsin and trypan blue were from GIBCO Laboratories (Grand Island, NY).
MEM with folate (2 µmol/L), thymidine (36 µmol/L), hypoxanthine (36 µmol/L), glycine (600 µmol/L), serine (250 µmol/L) and 10% fetal bovine serum. Penicillin (20,000 units/L), streptomycin (20 mg/L) and fungizone (2.5 mg/L) were also added to media for the elimination of contamination. To formulate folate-deficient media, folate as well as thymidine, hypoxanthine and glycine were omitted from complete media to stress substrate availability in one carbon metabolism. To minimize exogenous folate sources, fetal bovine serum was replaced with dialyzed fetal bovine serum (dFBS), which had been dialyzed at 4°C for 16 h against 6 × 10 volumes of sterile PBS. Control medium was complete media with 10% dFBS. Therefore, HepG2 cells cultured in folate-deficient medium are designated as folate-deficient cells. HepG2 cells cultured in control medium are referred to as control cells.
. Briefly, cells from control or folate-deficient cultures were trypsinized, and intracellular folate was extracted by extraction buffer [20 g/L sodium ascorbate, 50 mmol/L HEPES, 50 mmol/L CHES (2-[N-cyclohexylamino]-ethanesulfonic acid), 1.4% 
-mercaptoethanol, pH 7.85]. Upon treatment with chicken pancreas conjugase (v/v: 4:1), total intracellular folate content was quantified by microbiological assay using glycerol-protected L. casei (ATCC no. 7469) in 96-well microtiter plates. Absorbance was measured at 600 nm in a MRX model ELISA reader (Dynatech Laboratories, West Sussex, UK).
. Cells cultured at each week of folate deficiency were harvested, fixed in ice-cold 100% ethanol and treated with ribonuclease A (500 mg/L) and 0.5% Triton at 37°C for 60 min. Cells were then stained with propidium iodide (PI: 50 mg/L for DNA staining) for 20 min. After centrifugation, the pellet was resuspended in PBS with the same concentration of PI and fluorescein isothiocyanate (FITC: 0.25 mg/L for protein staining) for 10 min. Cellular DNA and protein contents in 10,000 cells were analyzed in a FACScan flow cytometer using ModFit LT 2.0 software program (Becton Dickinson, San Jose, CA). The percentage of cells in each phase of the cell cycle was determined by CELL Quest program (Becton Dickinson). Protein contents were also measured using the Bio-Rad protein assay (Bradford 1976). A standard curve was prepared with several dilutions of bovine serum albumin ranging from 4 to 24 g/L when the assay was performed. The mixture was read at OD595 using the MRX model ELISA reader (Dynatech Laboratories).
). Thin sections were stained with 4% uranyl acetate and 0.2% lead citrate and examined on JEOL 200 EX II Electron Microscope (JEOL, Tokyo, Japan).
. Briefly, 3 × 106 of control or folate-deficient cells at the end of each week were suspended in ice-cold lysis buffer (100 mmol/L NaCl, 10 mmol/L Tris HCl, 25 mmol/L EDTA, 0.5% sodium dodecylsulfate and 0.3 g/L proteinase K) for 15 h in a 50°C water bath. DNA was extracted by phenol/chloroform/isoamyl alcohol method (25:24:1). After 100% alcohol precipitation, DNA was dissolved in 10 mmol/L Tris-HCl and 1 mmol/L EDTA (pH 8.0). RNA was digested with 1 mg/L ribonuclease A for 1 h at 37°C. DNA fragments were electrophoresed on a 2% agarose minigel at 100 V for 40 min and visualized with ethidium bromide staining under UV illumination. Multimers of 100 base pair DNA were used as DNA markers (Pharmacia Biotech, Piscataway, NJ).
. In Situ Cell Death Kit (Boehringer Mannheim GmbH Biochemica, Germany) was used. Aliquots of 2 × 106 cells were harvested from control or folate-deficient cultures and fixed in 4% buffered paraformaldehyde (pH 7.4) for 30 min at room temperature. After washing in PBS, cells were resuspended in permealization solution (0.1% Triton and 0.1% sodium citrate) for 2 min at 4°C. The cells were washed twice and incubated with the TUNEL reaction mixture at 37°C for 60 min. TUNEL reagents contained terminal deoxynucleotidyl transferase from calf thymus and fluorescein dUTP. DNA of fixed cells labeled by the addition of fluorescein dUTP at strand breaks by terminal transferase was measured with a FACScan flow cytometry (Becton Dickinson). TUNEL positive cells were analyzed and gated by CELL Quest program (Becton Dickinson).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
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Fig 1.
Relative intracellular folate concentrations and growth rates (A) and cellular viability of HepG2 cells (B) grown in control or folate-deficient culture media. Data plotted are means ± SD, n = 6. Death rates of folate-deficient cells at wk 2-5 were significantly greater than those of the control as evaluated by Student's paired t test (P < 0.05).
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Fig 2.
Agarose gel electrophoresis of DNA from HepG2 cells cultured in the complete, control or folate-deficient media. Lane M: DNA markers of molecular size at 100 base pair multimers. Lane 0: DNA from HepG2 cells cultured in complete media. Lanes 1-4: DNA extracted from plate-adherent HepG2 cells cultured in control or folate deficient media for 1, 2, 3 and 4 wk as indicated.
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Fig 3.
Transmission electron microphotographs of nuclear chromatin condensation of HepG2 cells cultured in control or folate-deficient media for 2 wk. Magnification: 5000×. Nuclear chromatin condensation is marked by an arrow. Bar, 1 µm.
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Fig 4.
Cell cycle analysis of HepG2 cells cultured in control or folate-deficient media. (A) DNA frequency distribution histograms of control or folate-deficient cells in one representative sample. (B) The percentage of the cell population in G0/G1 phase (top), S phase (middle) or G2/M phase (bottom) in HepG2 cells cultured in control or folate-deficient medium for 1-4 wk. Values are means ± SD, n = 3. *Significantly different from the control (Student's paired t test; P < 0.05).
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Fig 5.
Effect of folate deficiency on protein content of HepG2 cells. (A) FITC (fluorescein isothiocyanate) fluorescence histograms of cellular protein content of HepG2 cultured in control or folate-deficient media as indicated. M represents the mean green fluorescence intensity of fixed cells stained with FITC from respective cultures in one representative experiment. Increase of M value denoted the higher mean protein content in culture cells. (B) Protein content of HepG2 cells cultured in control or folate-deficient medium was quantified using the Bio-Rad protein assay. Data are expressed as means ± SD, n = 6. *Significantly different from the control (Student's paired t test; P < 0.05).
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Fig 6.
Quantitative p53 levels in HepG2 cells cultured in control or folate-deficient medium. The mean p53 protein levels in the samples were analyzed by sandwich enzyme immunoassays (pantropic p53 quantitative ELISA). Data are expressed as means ± SD, n = 3. Values obtained from each folate-deficient condition were not significantly different from the control (Student's paired t test; P < 0.05).
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Fig 7.
Effect of folate supplementation on apoptotic folate-deficient HepG2 cells. (A) Percentage of TUNEL-positive cells after supplementation of 2 µmol/L folate to 3 wk folate-deficient HepG2 cells for 7, 18 and 23 d. Data are averaged value from duplicate cultures. (B) Changes of S phase cell population after supplementation of 2 µmol/L folate to 3 wk folate-deficient cells for 7, 18 and 23 d. Data plotted are expressed as means ± SD.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The data indicated that folate is necessary to support normal cellular proliferation and that folate deficiency leads to apoptotic cell death in HepG2 cells. Our findings are consistent with other studies reporting more apoptosis under nutritional folate-deficient conditions. Folate-deficient culture (22-24 h) resulted in apoptosis of proerythroblasts obtained from folate-deficient mice infected with Friend leukemia virus (Koury and Horne 1994). Apoptotic DNA fragmentation occurred in a dead Chinese hamster ovary cell line within 8 d of folate-deficient culture (James et al. 1994). Apoptotic bodies were detectable in histological preparations of livers from F344 rats fed folate and methyl donor-deficient diets (James et al. 1997). Taken together, our and others' studies suggest that apoptosis is the specific cell death mode through which localized folate-deficient cells would be eliminated.
). These results suggest that apoptosis of HepG2 cells induced by folate deficiency is specific for S- and/or G2-phase arrest. Apoptosis triggered by inhibition of S-phase (and G2-phase) is often observed in response to the suppression of DNA replication or DNA repair in drug-induced cell death. Inhibitors of DNA topoisomerase I (camptothecin) or II (teniposide) preferentially triggered HL-60 cells into S phase where they were selectively susceptible to apoptosis (Del Bino and Darzynkiewicz 1991, Gorczyca et al. 1993). The anticancer prodrug agent, mafosfamide, hindered DNA replication by alkylating DNA. It promoted apoptosis of HL-60 cells in S- and G2-phase arrest (Davidoff and Mendelo 1993). Another anticancer drug, 5-fluorouracil, which causes deoxyribonucleoside triphosphate (dNTP) pool imbalance and results in subsequent breakdown of DNA synthesis or impairment of DNA repair, induced apoptosis with G2 phase block in human breast cancer grafted in nude mice (Okamoto et al. 1996). Folate-mediated one carbon metabolism is essential for the de novo synthesis of both purines and the pyrimidine thymidylate. Deficiency of folate (or vitamin B-12) reduces the rate of DNA strand replication in phytohemagglutinin-stimulated lymphocytes derived from patients with megaloblastic anemia (Wickremasinghe and Hoffbrand 1980). In folate-deficient HepG2 cells, growth inhibition and S-phase arrest precede apoptosis, suggesting that the block of DNA replication and/or mitosis may be involved in apoptosis induction. Whether the decreased DNA synthesis and/or the limited capacity of DNA repair are attributable to the cell cycle-specific apoptosis of folate-deficient HepG2 cells, however, requires further investigation.
, El-Deiry et al. 1993, Xiong et al. 1993) or triggers apoptosis (reviewed in Yonish-Rouach 1996). In HepG2 cells, exposure to divergent stimuli such as cyclohexamide or staurosporine elevated p53 levels with the concomitant induction of apoptosis (Jiang et al. 1996). On the contrary, we demonstrated that the apoptotic response of HepG2 cells induced by folate deficiency was not dependent upon p53, since the depletion of this essential nutrient did not affect p53 protein levels (Fig. 6). Our finding is in agreement with another report that there was no increase in p53 protein levels as assessed by Western blot analysis in liver from folate and methyl-deficient rats (Christman et al. 1993). Moreover, DNA breaks within the p53 gene were found in the same animal model (Pogribny et al. 1995b), suggesting that signaling pathways of apoptosis in nutritional folate deficiency may be distinctive from those in pharmacological cell death through p53-dependent manner.
). Selective inhibition of thymidylate synthesis to block DNA synthesis by the folate antagonist, methotrexate, caused unbalanced cellular events in the growth of cultured human leukemic cells (Taylor and Tattersall 1981). Methotrexate treatment induced DNA fragmentation in human leukemic cells (Kaufman 1989). In this present study, mean protein levels in HepG2 cells progressively increased (Fig. 5A), suggesting that protein synthesis in HepG2 cells was not suppressed under this particular condition. When coupled with S phase arrest, continuation of protein synthesis may partially account for the induction of apoptosis in folate-deficient HepG2 cells, although we do not know if inhibition of protein synthesis in this case would suppress apoptotic progression.
). dNTP pool imbalance may be considered to cause the failure of DNA replication and/or repair and leads to mutagenesis (Kunz and Kohalmi 1990). Folate deficiency induced dNTP imbalance in lymphocytes (James et al. 1994) and Chinese hamster ovary cell lines (James et al. 1994). Apoptosis in combination with dNTP imbalance was also observed in a Chinese hamster ovary cell line (James et al. 1994) and in the livers of rats fed folate and methyl-deficient diet (James et al. 1997). Our preliminary study has revealed that dNTP imbalance may be responsible for the cell cycle phase-specific apoptosis in folate-deficient HepG2 cells. The role of dNTP imbalance to signal the induction of apoptosis in HepG2 cells is currently under investigation in our laboratories.
). High folate contents in hepatocytes may protect them from rapid folate depletion. Higher intracellular folate levels are observed in HepG2 cells compared to those of Chinese hamster ovary cells (Borman and Branda 1989). Second, cellular doubling time has been suggested to be critical for intracellular folate turnover and the onset of functional folate deficiency (Steinberg et al. 1983). HepG2 cells exhibited longer doubling time (29 ± 3 h) than Chinese hamster ovary cells (12 h). Third, high affinity folate receptors may be present or be induced in folate-deficient HepG2 cells since HepG2 cells can survive in media of very low folate concentrations (5 nmol/L) (Hsu 1992, Huang and Lin 1997). In several malignant cell lines, high concentrations of membrane folate-binding receptors have been found that enable the cells to proliferate at very low folate medium levels (Kamen and Capdevila 1986, Matsue et al. 1992). Further studies are needed to characterize folate-mediated one carbon metabolism and associated folate receptors mechanism in HepG2 cells.
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FOOTNOTES |
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Manuscript received 8 July 1998. Initial reviews completed 23 July 1998. Revision accepted 11 September 1998.
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ACKNOWLEDGMENTS |
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The authors thank Hsiu-Yuan Liao for technical assistance with Transmission Electron Microscopy and Li-Tun Huang for assistance with the flow cytometer at Chang Gung University. We acknowledge Mei-Kwei Yang and Yung-An Lee in the Department of Biology at Fu-Jen University for providing UV illumination equipment to photograph the agarose minigels.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-irradiation in Chinese hamster ovary cells.
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