Cell death in cancer and development. AACR special conference in cancer research.
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Apoptosis↗
Biomedical subjects
A Eastman
Publications and source records attributed to A Eastman.
Enhancement of sensitivity to platinum(II)-containing drugs by 12-O-tetradecanoyl-phorbol-13-acetate in a human ovarian carcinoma cell line.
Sensitivity to platinum-containing drugs is believed to be a function of how much drug enters the cell, the extent of DNA adduct formation and the rate at which DNA is repaired. Activation of protein kinase C by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was found to enhance the sensitivity of human ovarian carcinoma 2008 cells to cisplatin (DDP), carboplatin (CBDCA) and (glycolato-O,O') diammineplatinum(II) (254-S). TPA was able to enhance the sensitivity of the DDP-resistant 2008/C13*5.25 subline to each of the three drugs to the same extent as for the 2008 cells. TPA produced no significant change in the uptake of [3H]cis-dichloro(ethylenediamine)-platinum(II). ([3H]DEP) or CBDCA. It did not alter glutathione content or glutathione-S-transferase activity, and induced rather than suppressed metallothionein IIA mRNA levels. TPA did increase the formation of intrastrand guanine-guanine cross-links by a factor of 1.5 +/- 0.3 (s.d.), and reduced the fraction of intrastrand adducts removed from DNA over the subsequent 24 h by a factor of 1.3 +/- 0.2 (s.d.) (n = 4; P < 0.05), however, these effects were too small to account for the degree of TPA-induced sensitisation. These results indicate that the mechanism of TPA-induced sensitisation is not specific to any one structural form of platinum-containing drug, and that it is not readily explicable on the basis of an effect on the four major parameters currently believed to regulate DDP sensitivity.
Carboplatin↗
Deoxyribonuclease II in apoptosis and the significance of intracellular acidification.
Many endonucleases have been identified in cells, but which are involved in apoptosis remains controversial. We detected and characterized an endonuclease as deoxyribonuclease II. Its most important characteristic is its acidic pH optimum that requires decreased intracellular pH for activation. Intracellular acidification has been observed during apoptosis in a number of systems. This acidification results from a selective loss of pH regulation, and is likely due to dephosphorylation of proton exchangers. The fact that growth factors normally prevent apoptosis and also phosphorylate ion exchangers suggests the critical role of intracellular kinase cascades for preventing apoptosis.
Journal Article↗
The role of cell cycle progression in cisplatin-induced apoptosis in Chinese hamster ovary cells.
Many anticancer drugs arrest cells in G2 of the cell cycle and subsequently induce cell death by apoptosis. The current experiments establish a detailed sequence of events that occur in Chinese hamster ovary CHO/UV41 cells following incubation with cisplatin. Synchronized CHO/UV41 cells were damaged with cisplatin in early S. The cells progressed at a normal rate through S but arrested in G2. The arrested cells exhibited normal levels of the mitosis-promoting kinase p34cdc2 in its fully phosphorylated, inactive form. After a protracted arrest, the cells dephosphorylated p34cdc2 and underwent an aberrant mitosis and cytokinesis in which the chromosomes segregated unequally due to the formation of multipolar mitotic spindles. These cells subsequently lost contact with the extracellular matrix, and only then digested their DNA in a manner characteristic of apoptosis. This sequence of events could be dramatically accelerated by the addition of caffeine to G2-arrested cells, which induced dephosphorylation of p34cdc2 and passage through an aberrant mitosis. It has previously been suggested that protein synthesis is required for both caffeine-induced premature mitosis and apoptosis. However, when added in G2, cycloheximide could inhibit neither the caffeine-induced mitosis nor apoptosis. Inhibition was only seen if cycloheximide was added during S before complete synthesis of the proteins required for mitosis. These results demonstrate that, in this model, the proteins thought to be involved in apoptosis are those required for normal cell cycle progression. It is hypothesized that the DNA digestion results from loss of signal transduction originating from the extracellular matrix but that earlier events leading to loss of cell adhesion are critical for the induction of apoptosis.
Animals↗
Etoposide-induced apoptosis in human HL-60 cells is associated with intracellular acidification.
Apoptosis is a pathway of cell death characterized by internucleosomal digestion of genomic DNA. Such DNA digestion can be induced by both physiological stimuli and cytotoxic treatment with many anticancer agents. This digestion has generally been considered to be mediated by a Ca2+/Mg(2+)-dependent endonuclease that is activated by increases in intracellular Ca2+. However, we suggest that an alternate endonuclease, DNase II, may be a more likely candidate. In these studies, apoptosis was induced in human HL-60 cells by a 30-min incubation with the topoisomerase II inhibitor etoposide. DNA digestion characteristic of apoptosis began within 3 h of removal of etoposide. Morphological indication of apoptosis was observed concurrently. Only about 20% of the cells underwent apoptosis at this time; these appeared to be cells in S phase at the time of etoposide treatment. The remainder of the cells progressed to the G2 phase and arrested there for at least 48 h. Intracellular Ca2+ and pH were measured in individual cells by flow cytometry. No changes in intracellular Ca2+ were observed, but an acidification of up to 1 pH unit occurred in about 15% of the cells and correlated with the time course of appearance of DNA digestion. Cells were sorted on the basis of intracellular pH and only the acidic cells showed the morphology and DNA digestion characteristic of apoptosis. These results demonstrate the involvement of DNase II in apoptotic DNA digestion and suggest mechanisms of pH homeostasis as regulators of apoptosis.
Apoptosis↗
Kinetic and DNA-binding properties of recombinant human O6-methylguanine-DNA methyltransferase.
O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that plays an important role in chemotherapy, mutagenesis, and carcinogenesis. Recombinant human MGMT was isolated from an Escherichia coli high performance expression system and purified to homogeneity. The kinetic and DNA-binding properties of the recombinant human MGMT were studied. The purified human MGMT reacted stoichiometrically with methylated DNA under second-order rate kinetics. The rate constant with normal methylated DNA was 1 x 10(9) M-1 min-1 at 37 degrees C. The binding to DNA was the rate determining step in the repair process. Approximately eight base pairs of the DNA substrate were covered by the human MGMT protein. The affinity constant for interaction of DNA to MGMT was approximately 4.7 x 10(5) M-1. The binding to methylated DNA was also examined; the binding affinity to methylated DNA was two times higher than that to unmodified DNA. The interaction with DNA induced a conformational change in the human MGMT protein as monitored by circular dichroism and fluorescence analysis. A similar conformational change was induced by both methylated and unmodified DNA.
Base Sequence↗
Identification of deoxyribonuclease II as an endonuclease involved in apoptosis.
Cell death occurs by apoptosis during programmed deletion of cells and following exposure to cytotoxic agents. Central to the mechanism of apoptosis is internucleosomal DNA digestion by an endogenous endonuclease which is thought to mediate cell death. An axiom of apoptosis is that the endonuclease involved is a Ca2+/Mg(2+)-dependent endonuclease. During purification of endonucleases from Chinese hamster ovary cells, we found little Ca2+/Mg(2+)-dependent endonuclease activity, but large amounts of an endonuclease active below pH 7. This acidic endonuclease was activated in intact cells by reducing intracellular pH values below 7 with a proton ionophore. This activity generated internucleosomal digestion of DNA characteristic of apoptosis. Nuclear extracts contained a cation-independent endonuclease with identical pH-dependent activity. We have compared the acidic endonuclease to bovine deoxyribonuclease II (DNase II) and have found them nearly identical by all tests, including sensitivity to various inhibitors, purification by the same chromatographic steps, and recognition by antibody raised against the bovine enzyme. Addition of either the acidic endonuclease or bovine DNase II to isolated nuclei induced internucleosomal DNA digestion up through pH 6.5. These data demonstrate that DNase II can mediate internucleosomal DNA digestion characteristic of apoptosis following intracellular acidification. Furthermore, these data question the premise that the Ca2+/Mg(2+)-dependent endonuclease is the only endonuclease involved in apoptosis.
Acid Phosphatase↗
Dietary fish oil reduces microthrombi over atherosclerotic lesions in hyperlipidemic swine even in the absence of plasma cholesterol reduction.
We have investigated in swine the effect of fish oil additives to a butter-cholesterol hyperlipidemic diet (BT) on atherogenesis and thrombogenesis when average plasma cholesterol levels were kept similar in fish oil-treated and untreated BT groups. The studies included evaluation of lesion sizes and cell numbers, counts of adherent monocytes over lesions, and counts of platelet clumps (microthrombi) over lesions either attached directly to endothelium or to adherent monocytes. Anatomic sites studied for lesion development were the left anterior descending coronary artery (LAD), the distal 1/5 of the abdominal aorta, and a proximal portion of the thoracic aorta. Counts of attached monocytes and platelet clumps were made by scanning electron microscopy only for the LAD and expressed per mm2 of surface. The most striking new result was in regard to the platelet clumps. These were reduced by the fish oil from 996 +/- 295/mm2 in the untreated BT group to 313 +/- 59 and 364 +/- 105 in BT+cod liver oil and BT+menhaden oil groups, respectively. Most of the platelet clumps were adherent to attached monocytes in all groups and the number of attached monocytes were greatly reduced by the fish oil additive. Thus there were close relationships among platelet clumps, monocytes, and lesion endothelium. Numbers of attachments over nonlesion endothelium were much less than those over lesions in all dietary groups. The most surprising result was the lack of retardation of lesion growth by the fish oil additives in spite of the reduction in attached monocytes and platelet clumps. In previous studies where the high plasma cholesterol levels in the BT swine had been modestly reduced (about 25%) there had been a marked retardation of lesion growth. The current result suggests that plasma cholesterol is the major factor controlling lesion growth in this model through under milder conditions and longer observation periods other factors might become apparent.
Animals↗
Apoptosis: a product of programmed and unprogrammed cell death.
Apoptosis is a form of cell death defined by morphological and biochemical characteristics. Although originally described in 1972, it is only very recently that significant interest in the subject has occurred, possibly as a result of the identification of genes that may either positively or negatively regulate the process. With the rapid expansion of knowledge, it has become apparent that there are multiple pathways that induce apoptosis; some of these may represent programmed events but others are clearly unprogrammed. To clarify the terminology used, it is recommended that apoptosis be used as originally defined to refer only to the end product of these pathways. Furthermore, the realization that a cell can die by multiple pathways suggests caution when translating experimental results. Many potential intermediates may be identified but they may represent components of different pathways. Concern for the use of various inhibitors of apoptosis is also presented. Future directions will be aimed at the definitive identification of the signal mechanisms regulating apoptotic cell death.
Animals↗
Gene-specific damage produced by cisplatin, ormaplatin and UV light in human cells as assayed by the polymerase chain reaction.
The level of DNA damage in the total genome of leukocytes from patients receiving cisplatin therapy has been previously correlated with therapeutic outcome. Based on the fact that unrepaired damage in transcribed genes has been well correlated with sensitivity to a number of DNA damaging agents, the correlations with therapeutic response might be improved if the level of DNA damage in specific genes was assessed. We previously established a polymerase chain reaction (PCR)-based assay to measure cisplatin lesions in specific genes. The major advantages of this assay include its potential to quantify DNA lesions produced by any DNA damaging agent, and to measure it in minute samples of cells. This assay has been further improved to realize this potential. Specifically, cells were damaged with cisplatin, a related analogue ormaplatin, and ultraviolet light, and PCR was performed on the DNA from 1000 cells after direct lysis with no purification. Human HL-60 cells were used to compare the efficacy of the various agents to damage the alpha-fetoprotein gene. This was then extrapolated to damage in the total genome and compared to the cytotoxicity for each agent. At a dose that killed 50% of the cells, ormaplatin, cisplatin and ultraviolet light produced 19,200, 48,000, and 1,080,000 lesions per cell, demonstrating that ormaplatin lesions were the most effective at killing cells. Mononuclear cells were obtained from freshly isolated blood from five individuals. These cells were damaged with the three agents, and PCR of the alpha-fetoprotein gene was performed. No significant difference between the individuals was observed in the level of DNA damage produced by any of the agents.(ABSTRACT TRUNCATED AT 250 WORDS)
Antineoplastic Agents↗
Modulation of cisplatin resistance in human malignant melanoma cells.
Previous studies have shown that the combination of dacarbazine, carmustine, cisplatin (DDP), and tamoxifen (TAM) produced a 53% overall response rate in patients with disseminated melanoma. Deletion of TAM from the regimen resulted in a fall in the response rate to 10%, and reincorporation of TAM returned the response rate to 52%, suggesting an important role for TAM. Using the human melanoma cell line T-289, we examined the nature of the interaction between TAM and each member of this combination in clonogenic assays in soft agar. The combination of TAM with DDP was highly synergistic as demonstrated by median effect analysis, whereas TAM was antagonistic with carmustine and an activated form of dacarbazine. The mean combination index at 50% kill was 0.26 +/- 0.02 (mean +/- SD) for TAM and DDP. This marked synergism was observed at concentrations of TAM that are clinically achievable. TAM had no effect on the uptake of the DDP analogue [3H]dichloro(ethylenediamine)platinum(II). There was no effect on the formation or repair of DDP intrastrand DNA adducts. Similarly, there was no effect demonstrated on the intracellular concentrations of glutathione or metallothioneins. We conclude that the interaction between TAM and DDP is truly synergistic in this cell line and is accomplished through none of the four mechanisms commonly associated with DDP resistance.
Carboplatin↗
Endonuclease activation during apoptosis: the role of cytosolic Ca2+ and pH.
An axiom of apoptosis is that increases in cytosolic Ca2+ activate a Ca2+/Mg(2+)-dependent endonuclease. However, when HL-60 human promyelocytic leukemia cells were incubated with the Ca2+ ionophore ionomycin in varied extracellular Ca2+, DNA digestion was independent of extracellular Ca2+. Under these conditions, intracellular Ca2+ concentrations did not correlate with the observed DNA digestion. In contrast, intracellular acidification correlated well with DNA digestion. These data indicate that increased intracellular Ca2+ is not the primary signal for endonuclease activation in all forms of apoptosis, but that intracellular acidification may be involved. The observed intracellular acidification is consistent with the involvement of deoxyribonuclease II in apoptosis.
Calcium↗
Irradiation-induced expression of O6-methylguanine-DNA methyltransferase in mammalian cells.
O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein which plays an important role in chemotherapy, mutagenesis, and carcinogenesis. The specific activity of MGMT in female rat liver can be induced by approximately 20-fold by treatment of the rats with gamma-irradiation. Maximum response occurred 48 h after 15 Gy irradiation. MGMT levels in male rats were induced by only 3-fold. MGMT activity was also induced by irradiation of rat hepatoma H4IIE cells with a 3-fold increase noted after treatment with 3 Gy. Northern analysis and nuclear run-on assays indicated that the induction of MGMT was regulated at the transcriptional level. The radiation-mediated increase in MGMT was blocked by H7, a protein kinase inhibitor, but not by H89, an inhibitor of protein kinase A. Hydroxyl radicals may play a role in the induction mechanism since dimethyl sulfoxide, a radical scavenger, blocked the radiation-mediated increase in MGMT. MGMT activity was also increased by treatment of the cells with H2O2, in accordance with the involvement of activated oxygen species in the induction of MGMT. Finally, the addition of cycloheximide, an inhibitor of protein synthesis, prior to but not after irradiation, abolished the increase in MGMT activity.
Animals↗
Expression of human O6-methylguanine-DNA methyltransferase in a DNA excision repair-deficient Chinese hamster ovary cell line and its response to certain alkylating agents.
A plasmid has been constructed in which the expression of human O6-methylguanine-DNA methyltransferase (MGMT) complementary DNA is driven by the Rous sarcoma virus promoter sequence. We had previously shown that transfection of this plasmid into Chinese hamster ovary (CHO) cells results in the expression of MGMT and in increased cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine and 1-(2-chloroethyl)-1-nitrosourea (CNU) but not N-nitroso-N-ethylurea (ENU). In the present study, the Rous sarcoma virus promoter-MGMT was transfected into DNA excision repair-deficient CHO UV41 cells to investigate the phenotype associated with MGMT expression in the absence of DNA excision repair. Both the UV41/MGMT and CHO/MGMT cells expressed similar levels of MGMT and exhibited a similar increased resistance to N-methyl-N'-nitro-N-nitrosoguanidine. The UV41 cells were 20-fold more sensitive to CNU than the wild-type CHO cells. Expression of MGMT increased the resistance to CNU about 6-fold in both cell lines, but the difference between the two cell lines attributable to the excision repair defect still persisted. The UV41 cells were 2- to 3-fold more sensitive than the wild-type CHO cells to the monofunctional alkylating agents 1-(2-hydroxyethyl)-1-nitrosourea and ENU, but the MGMT phenotype did not alter sensitivity. This suggests that alkylation at the O6 position of guanine has no role in cytotoxicity of ethylating agents and that monofunctional DNA damage has little role in the cytotoxicity of CNU. Since MGMT can prevent the formation of G-C interstrand cross-links formed by CNU, other excision repair-sensitive DNA adducts must play a major role in the sensitivity of UV41 cells to this bifunctional alkylating agent. These results suggest that DNA intrastrand cross-links may be major contributors to the cytotoxicity of CNU.
Animals↗
Modulation of cisplatin sensitivity and growth rate of an ovarian carcinoma cell line by bombesin and tumor necrosis factor-alpha.
A twofold change in the cisplatin (DDP) sensitivity of 2008 human ovarian carcinoma cells is sufficient to reduce tumor response in vivo. The DDP sensitivity of these cells can be enhanced by activation of the epidermal growth factor and protein kinase C signal transduction pathways. We report here that two endogenous growth factors, bombesin and tumor necrosis factor alpha (TNF alpha), enhanced DDP sensitivity by factors of 1.7 +/- 0.1 (SD)-fold and 1.8 +/- 0.1 (SD)-fold, respectively. Both agents also produced sensitization in an 11-fold DDP-resistant 2008 subline. Neither bombesin nor TNF alpha changed the accumulation of DDP, glutathione content, or glutathione-S-transferase activity in 2008 cells. However, a 2-h exposure to both bombesin and TNF alpha was sufficient to increase 2008 cloning efficiency by up to 2.6 +/- 0.1 (SD)-fold and 2.2 +/- 0.1 (SD)-fold, and it increased average colony size by 1.35 +/- 0.1 (SD)-fold and 1.55 +/- 0.1 (SD)-fold, respectively. Bombesin increased intracellular free calcium, and this was blocked by the bombesin receptor-specific antagonist SC196, demonstrating that 2008 cells have functional bombesin receptors. These results indicate that bombesin and TNF alpha can enhance sensitivity to DDP in both DDP sensitive and resistant variants of a human ovarian carcinoma and that both agents serve as growth factors for this tumor.
Bombesin↗
The origins of DNA breaks: a consequence of DNA damage, DNA repair, or apoptosis?
DNA breaks can arise from many sources after incubation of cells with toxic agents. Very few agents break DNA directly, rather most breaks occur as a result of metabolic participation by the cell, such as during attempts to repair the damage. It is now realized that many DNA breaks arise as a consequence of steps in the pathway of cell death. Upon reanalyzing the methodology commonly used to detect DNA breaks, it is evident that many studies would not have observed DNA breaks associated with cell death. Frequently experimental conditions have been used that are extremely toxic to cells with the justification that the cells were still viable as measured by their ability to exclude dyes such as trypan blue. However, the DNA digestion associated with cell death by apoptosis occurs prior to changes in membrane integrity. Because the possibility of endogenous endonuclease activity was not realized, many studies may have inaccurately assumed that DNA breaks arose during, for example, inhibition of DNA repair or as intermediates in recombination. In light of the new understanding of apoptosis and the formation of DNA breaks as an early event in cell death, it is important to both reevaluate past conclusions and to ensure that future studies fully consider the breaks derived from the cytotoxicity of every agent under investigation.
Animals↗
A polymerase chain reaction-based method to detect cisplatin adducts in specific genes.
Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125 microM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 microM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.
Animals↗
Expression of the c-Ha-ras oncogene in mouse NIH 3T3 cells induces resistance to cisplatin.
The effect of expression of the c-Ha-ras oncogene on cisplatin (DDP) sensitivity was examined in murine NIH 3T3 cells transfected with the dexamethasone (DEX)-inducible mouse mammary tumor virus promoter linked to an activated c-Ha-ras gene [LTR H-ras(A) cells]. Treatment of these cells with 5 microM DEX for 24 h induced c-Ha-ras expression and produced an 8.2 +/- 1.3-fold (SD) increase in DDP resistance as quantitated by clonogenic assay. Induction of the c-Ha-ras oncogene reduced DDP accumulation by 40% and intrastrand adduct formation by 17%. In nontransfected wild-type NIH 3T3 cells, DEX did not induce DDP resistance nor did it decrease DDP accumulation. Induction of c-Ha-ras expression did not alter cellular glutathione content or the activity of glutathione-S-transferase in the LTR H-ras(A) cells. DEX increased cellular metallothionein content by 1.6-fold in NIH 3T3 cells and 3.3-fold in LTR H-ras(A) cells. We conclude that DEX-induced overexpression of a mutant c-Ha-ras gene confers DDP resistance and that this resistance is associated with an impairment of cellular drug accumulation and an increase in metallothionein content.
3T3 Cells↗