Cell damage-induced conformational changes of the pro-apoptotic protein Bak in vivo precede the onset of apoptosis.

Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xL and Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xL dissociation from Bak occurred on exposure of Bak's NH2 terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death.

Positive regulation of apoptosis by HCA66, a new Apaf-1 interacting protein, and its putative role in the physiopathology of NF1 microdeletion syndrome patients.

As a component of the apoptosome, a caspase-activating complex, Apaf-1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. We report here the identification of a novel Apaf-1 interacting protein, hepatocellular carcinoma antigen 66 (HCA66) that is able to modulate selectively Apaf-1-dependent apoptosis through its direct association with the CED4 domain of Apaf-1. Expression of HCA66 was able to potentiate Apaf-1, but not receptor-mediated apoptosis, by increasing downstream caspase activity following cytochrome c release from the mitochondria. Conversely, cells depleted of HCA66 were severely impaired for apoptosome-dependent apoptosis. Interestingly, expression of the Apaf-1-interacting domain of HCA66 had the opposite effect of the full-length protein, interfering with the Apaf-1 apoptotic pathway. Using a cell-free system, we showed that reduction of HCA66 expression was associated with a diminished amount of caspase-9 in the apoptosome, resulting in a lower ability of the apoptosome to activate caspase-3. HCA66 maps to chromosome 17q11.2 and is among the genes heterozygously deleted in neurofibromatosis type 1 (NF1) microdeletion syndrome patients. These patients often have a distinct phenotype compared to other NF1 patients, including a more severe tumour burden. Our results suggest that reduced expression of HCA66, owing to haploinsufficiency of HCA66 gene, could render NF1 microdeleted patients-derived cells less susceptible to apoptosis.

Analysis of cyclin B1 and CDK activity during apoptosis induced by camptothecin treatment.

We have studied the role of cyclins and cyclin-dependent kinase (CDK) activity in apoptosis induced by camptothecin (CPT). In this model, 22% of the cells stain for annexin-V at 24 h and then proceed to be 93% positive by 72 h. This time window permits the analysis of cyclins in cells that are committed to apoptosis but not yet dead. We provide evidence that cyclin protein levels and then associated kinase levels increase after CPT treatment. Strikingly, cyclin B1 and cyclin E1 proteins are present at the same time in CPT treated HT29 cells. Although cyclin B1 and E1 CDK complexes are activated in CPT treated cells, only the cyclin B1 complex is required for apoptosis since reduction of cyclin B1 by RNAi or roscovitine treatment reduces the number of annexin-V-stained cells. We have detected poorly organized chromosomes and phosphorylated histone H3 epitopes at the time of maximum cyclin B1/CDK kinase activity in CPT-treated cells, which suggests that these cells enter a mitotic catastrophe. Understanding which CDKs are required for apoptosis may allow us to better adapt CDK inhibitors for use as anti-cancer compounds.

Bid, a widely expressed proapoptotic protein of the Bcl-2 family, displays lipid transfer activity.

Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis in primary tissues such as liver. Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria to facilitate the release of apoptogenic cytochrome c. The mechanism of Bid relocation to mitochondria was unclear. We report here novel biochemical evidence indicating that Bid has lipid transfer activity between mitochondria and other intracellular membranes, thereby explaining its dynamic relocation to mitochondria. First, physiological concentrations of phospholipids such as phosphatidic acid and phosphatidylglycerol induced an accumulation of full-length Bid in mitochondria when incubated with light membranes enriched in endoplasmic reticulum. Secondly, native and recombinant Bid, as well as tBid, displayed lipid transfer activity under the same conditions and at the same nanomolar concentrations leading to mitochondrial relocation and release of cytochrome c. Thus, Bid is likely to be involved in the transport and recycling of mitochondrial phospholipids. We discuss how this new role of Bid may relate to its proapoptotic action.

Epigenetic determinants of resistance to etoposide regulation of Bcl-X(L) and Bax by tumor microenvironmental factors.

BACKGROUND: Epigenetic factors (i.e., alterations of gene activity not involving mutations), as well as genetic changes in surviving cancer cells, may play an important role in drug resistance following cancer chemotherapy-a common cause of tumor relapse. Bcl-2 family proteins are central to the regulation of apoptotic cell death and modulate drug sensitivity. We investigated how survival signals in the cellular microenvironment affect the expression, protein conformation, and protein-protein interactions of the Bcl-2 family proteins Bax and Bcl-x(L) and how changes in response to microenvironmental signals alter the response of cancer cells to the drug etoposide. METHODS: JLP119 human B-lymphoma cells were treated with etoposide (40 microM) and then cultured in the presence of an activating anti-CD40 antibody, vascular cellular adhesion molecule-1 (VCAM-1)-to activate VLA-4 (alpha4beta1) integrin, and interleukin 4. Cell fate was monitored after etoposide treatment with or without these microenvironmental signals. Bcl-x(L) gene transcription and protein levels of Bcl-x(L) and Bax were measured by northern and western blotting, respectively. Nuclear translocation of transcription factor NF-kappaB was monitored by immunofluorescence and inhibited by (E)-capsaicin. Bax conformation and Bax-Bcl-x(L) interactions were monitored by immunofluorescence and immunoprecipitation, respectively. RESULTS: Microenvironmental survival signals produced statistically significant reductions in etoposide-induced apoptotic cell death, from 84.6% (95% confidence interval [CI] = 76.7%-92.4%) to 21.3% (95% CI = 19.5%-23.0%); P<.001. Activation of surface protein CD40 increased Bcl-x(L) protein levels via an (E)-capsaicin-inhibitable activation of NF-kappaB; i.e. , (E)-capsaicin restored etoposide sensitivity. Interleukin 4 had no effect on Bcl-x(L) protein levels but accelerated the increase in Bcl-x(L) protein associated with CD40 activation. VCAM-1- and interleukin 4-mediated signals diminished conformational changes in Bax protein and prevented the etoposide-induced disruption of constitutive Bax-Bcl-x(L) binding. CONCLUSIONS: Microenvironmental factors reduce the sensitivity of a B-cell lymphoma to etoposide in vitro by modulating the expression and functions of Bax and Bcl-x(L). This interaction may provide a paradigm for epigenetically induced drug resistance in other tumors.

Toxin-induced increase in survival factor receptors: modulation of the threshold for apoptosis.

The threshold at which toxins induce cell death is thought to directly relate to the amount of injury sustained. We show that the threshold at which a cell initiates toxin-induced death may vary in response to changes in the trophic environment. Treatment of Rat-1 fibroblasts with 50-175 mM dimethylformamide (DMF) induced cell death by apoptosis. Addition of insulin-like growth factor 1 (IGF-1; 100 ng/ml) and/or overexpression of the IGF-1 receptor (IGF-1R) attenuated the cytotoxicity of DMF. Furthermore, 95-99% of cells were protected from DMF-induced apoptosis if cells were pretreated with platelet-derived growth factor (5 ng/ml) for 16 h before treatment with DMF in the presence of IGF-1. Platelet-derived growth factor induced the expression of IGF-1R mRNA. The ability of cells to proliferate and survive after a 24-h treatment with DMF was determined by colony formation; whereas treatment with concentrations of >130 mM DMF reduced cellular survival, exposure to concentrations of <130 mM unexpectedly increased the colony-forming ability of treated cells when compared to that of controls. Treatment of Rat-1 fibroblasts with 75 and 130 mM DMF induced IGF-1R mRNA as determined by reverse transcription-PCR analysis. Serum withdrawal also transiently increased the expression of IGF-1R mRNA in Rat-1 fibroblasts. These results show that cells can actively adapt to pathological and physiological stress by up-regulating receptors that provide signals for cellular survival. We suggest that the threshold for toxin-induced apoptosis is determined not only by the extent of cytotoxic damage but also by the trophic environment and the ability of a cell to modulate survival signals that attenuate toxicity.

Effects of the antitumor drug Adriamycin on human red blood cell discocyte-echinocyte transitions.

The antitumor drug Adriamycin, when preincubated with human red blood cells (discocytes) for 10 min, prevented the formation of echinocytes induced by the calcium ionophore A23187 in the presence of 0.2 mM calcium. The degree of protection was concentration dependent and was greater than 90% at 10 microM Adriamycin. Adriamycin did not interfere with the accumulation of calcium induced by a 5 microM concentration of the ionophore. Adriamycin reversed echinocyte morphology to the discocyte form in echinocytes which had been formed by adenosine triphosphate depletion but not those formed after treatment with A23187 and Ca2+. Its ability to protect against Ca2+-induced echinocyte formation contrasts with the failure of the local anesthetic procaine to exert such an effect, even at 45 mM (J. Palek et al., Blood, 50: 155-164, 1977), and this difference suggests that Adriamycin may not be acting simply as a chaotropic agent. This hypothesis was supported by the observation that Adriamycin alone did not induce a cup-form morphology in discocytes (stomatocytosis). Wheat germ agglutinin protection of echinocyte formation induced by calcium loading was reversed by 30 mM N-acetylglucosamine, which partially reversed the Adriamycin protection of echinocyte formation. However, desialylation of human red blood cells with Clostridium perfringens type V neuraminidase, while preventing the protection of echinocyte formation by wheat germ agglutinin, had no effect on the protection afforded by Adriamycin. This suggests that Adriamycin does not prevent echinocyte formation via binding to the sialic acid residues of the transmembrane protein glycophorin and that another mechanism or mechanisms are involved in its action to modulate morphological transitions of the red blood cell membrane.

Correlation between the molecular weight and potency of polar compounds which induce the differentiation of HL-60 human promyelocytic leukemia cells.

Structure-activity studies of a series of polar organic compounds, including N,N-dimethylformamide, N-methylformamide, and related ureas and acetamides, were performed with regard to their ability to promote the terminal differentiation of the human promyelocytic leukemia cell line HL-60 to granulocyte-like cells. Functional and morphological criteria were used to assess the percentage of differentiated cells which arose from their continuous incubation with different concentrations of each agent over a period of 96 h. All of the alkylformamides, alkylacetamides, and alkylureas tested were found to induce differentiation, regardless of structure. Inspection of the results showed that there was a linear relationship (r = -0.937) between the molecular weight of each compound and the logarithm of the concentration which was required to bring about the differentiation of the greatest number of cells, while viability was generally maintained at greater than 85%. Once established, this relationship was used to predict the potency of several polar solvents which were structurally unrelated to the formamides. For example, methanol, ethanol, and acetone were all inducers of differentiation with a potency predictable from their molecular weight alone. The terminal differentiation induced by all of the compounds was only accomplished by cells which were capable of replication prior to differentiation. At concentrations which prevented a single replication and brought about a fall in cell viability over 96 h, no differentiation was observed. A correlation was observed between the molecular weight of each compound and the logarithm of its concentration to bring about cytotoxicity without differentiation (r = -0.935), and the line was almost parallel to that defining the concentration required for optimal differentiation (slope values of -0.02126 and -0.02288). A poorer (r = -0.6654) correlation was found between the logarithm of the octanol-water partition coefficient and the logarithm of the concentration required for optimal differentiation, when the data for 12 of the polar organic compounds were analyzed. The results suggest that no special structural requirements are necessary for the alkylformamides, -acetamides, -ureas, and related compounds to induce the terminal differentiation of HL-60 cells to granulocyte-like cells, but that the activity of each compound could be predicted from their molecular weight. The concentrations required to induce differentiation were marginally lower than those which were cytostatic or cytotoxic, which suggested that a toxic threat to the cells was sufficient to induce differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Investigations of mechanisms of drug-induced changes in gene expression: N-methylformamide-induced changes in synthesis of the M(r) 72,000 constitutive heat shock protein during commitment of HL-60 cells to granulocyte differentiation.

HL-60 cells were treated with the differentiating agent N-methylformamide and early changes in gene expression and protein content were investigated. Analysis of protein synthesis had previously shown an early (< 12 h) fall in the synthesis of M(r) 70,000 heat shock proteins (F. M. Richards, A. Watson, and J. A. Hickman. Cancer Res., 48: 6715-6720, 1988). The changes have now been characterized in detail and their kinetics compared to those of the expression of the c-myc protein. Immunoanalysis, using antibodies to either the stress-inducible heat shock protein hsp70 (4G4) or a pan-M(r) 70,000 heat shock protein antibody (3A3), showed that there was a striking reduction in the levels of the constitutive heat shock protein hsc70 when cells were incubated continuously with 170 mM N-methylformamide. A reduction in the level of hsc70 RNA was observed within 3 h and continued thereafter. In contrast, transcription of the hsc70 gene was induced within 1-2 h, after which the rate returned to basal level. There were no significant changes in the rate of transcription of the stress-inducible heat shock proteins hsp70 or hsp90. When N-methylformamide was removed from the cells, prior to commitment to differentiation, the levels of hsc70 were reestablished, whereas after 36 h of treatment there was no recovery. Western blotting with an antibody to the c-myc protein showed this to fall to virtually undetectable levels by 3 h under the same conditions. The results suggest that the loss of hsc70, which may perform a protein chaperoning role, was mediated at both transcriptional and posttranscriptional levels of regulation and was an early event closely associated with the commitment of HL-60 cells to differentiation. The fall in hsc70 was not associated with alterations in the cell cycle, nor were the kinetics of the change suggestive of a relationship with the decrease in content of c-myc protein.

The relationships between p53-dependent apoptosis, inhibition of proliferation, and 5-fluorouracil-induced histopathology in murine intestinal epithelia.

The relationship between acute (<36 h) induction of apoptosis and longer-term (>72 h) intestinal histopathology was systematically investigated in vivo using p53 wild-type (+/+) and null (-/-) mice. Administration of the enterotoxin 5-fluorouracil (5-FU) at either 40 or 400 mg/kg to BDF1 mice induced an acute p53-dependent apoptosis in the crypts of both small intestine and midcolon. Although the amount of apoptosis was of the same order of magnitude at its peak (24 h) at both doses, only 400 mg/kg 5-FU brought about histopathological changes to the gut after 96 h, quantified as losses of crypt and villus cellularity. Only after the administration of 400 mg/kg 5-FU were mitotic index and DNA synthesis significantly suppressed in both small intestinal and midcolonic crypts at 24 h. This correlated with a prolonged, p53-dependent expression of p21waf-1/cip1. In p53 null (-/-) mice, significant reductions in both 5-FU-induced apoptosis and inhibition of cell cycle progression allowed retention of crypt integrity 96 h after 5-FU. These results show that quantitative measures of acute apoptosis in vivo may not accurately predict subsequent pathological changes in the gut. Rather, p53-dependent inhibition of cell cycle progression, together with cell loss by apoptosis, caused a loss of crypt integrity. Importantly, the tissue toxicity of 5-FU was genetically determined at a locus (p53) separate from that directly associated with drug action.

Hypersensitivity of human testicular tumors to etoposide-induced apoptosis is associated with functional p53 and a high Bax:Bcl-2 ratio.

Metastatic testicular cancers are curable, whereas bladder cancers and most other solid tumors are not. Cell lines derived from human testicular (GH, GCT27, and 833K) and bladder (RT4, RT112, and HT1376) tumors retain this differential chemosensitivity in vitro. We have investigated the hypothesis that differential sensitivity to chemotherapy is related to differences in the threshold of susceptibility to undergoing apoptosis. Sensitivity to etoposide was not directly related to the frequency of DNA strand breaks. DNA damage was on average 2-fold greater in the testicular than the bladder tumor cell lines; in contrast, the testicular tumor lines were 15-fold more sensitive to etoposide cytotoxicity than the bladder tumor lines (IC90 values of 19 +/- 6 versus 293 +/- 180 microM, respectively). Using equidamaging (550 rad equivalents) etoposide treatments, the percentage of cells that underwent drug-induced apoptosis was on average higher in the testicular tumor cell lines than the bladder tumor cell lines. The testicular tumor lines have two characteristics that could confer sensitivity to drug-induced apoptosis. First, they have functional p53: the product of the p53-dependent gene waf-1 was increased after etoposide treatment. Second, the testicular tumor lines expressed relatively high levels of the apoptosis-promoting protein Bax, but there was no expression of the suppressor of apoptosis Bcl-2. In contrast, only one of the three bladder cell lines (RT4) had functional p53, and all of the bladder lines had readily detectable levels of Bcl-2 and low levels of Bax. In the testicular cell lines, increases in p53 and p53-transactivated genes were associated with apoptosis but not arrest in G1. In contrast, in the bladder cell line (RT4), increases in p53 and Waf-1 were associated with both arrest in G1 and apoptosis. The differences in the ratio of Bax:Bcl-2 could contribute to the differential sensitivity of the two tumor types. However, in contrast to earlier reports, the ratio of Bax and Bel-2 was not perturbed by DNA damage.

Inhibition of human erythrocyte inositol lipid metabolism by adriamycin.

Incubation of human erythrocytes with Adriamycin prevented their morphological transition from discocytes to echinocytes when they were either depleted of ATP or loaded with calcium. This effect was dependent upon drug concentration and cell density. Adriamycin (10(-5) M) prevented, by greater than 90%, the echinocytosis of 10(7) cells/ml (S. B. Chahwala and J. A. Hickman, Cancer Res., 45: 4986-4989, 1985), and 5 X 10(-4) M prevented that of 10(9) cells/ml. There was a poor correlation between the effects of Adriamycin as a modulator of this morphological transition and its potency as an inhibitor of calmodulin. Using inside-out red blood cell vesicles, Adriamycin inhibited calmodulin dependent Ca2+ uptake with a 50% inhibitory concentration of 5 X 10(-4) M. Adriamycin thus differs from other amphipathic drugs, such as those of the phenothiazine class, where inhibition of calmodulin correlated well with effects on erythrocyte morphology (G. A. Nelson, M. L. Andrews, and M. J. Karnovsky, J. Cell Biol., 96: 730-735, 1983). After 12 h of ATP depletion, levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] extracted from 10(9) erythrocytes/ml fell by 57% and after 48 h they fell by 97%, changes which were coincident with a 100% transition of morphology to echinocytes. Adriamycin, 5 X 10(-4) M-1 X 10(-3) M, maintained 10(9) cells/ml in a discocyte morphology and maintained PtdIns(4,5)P2 levels at 60-70% of the time zero controls, independently of the size of the fall in PtdIns(4,5)P2 levels. The data suggested that Adriamycin inhibited a discrete pool of PtdIns(4,5)P2 which may be responsible for the maintenance of a discocyte morphology. Neomycin, 10(-3) M, had no effect on the ATP depletion-induced discocyte-echinocyte transition of 10(9) erythrocytes/ml or on the fall in PtdIns(4,5)P2 levels. Adriamycin, like neomycin, prevented the calcium-induced breakdown of erythrocyte membrane vesicle PtdIns(4,5)P2 to inositol trisphosphate (50% inhibitory concentration, 7 X 10(-4) M) but, unlike neomycin (50% inhibitory concentration, 4.25 X 10(-4) M) it was able to inhibit breakdown by 100% at higher concentrations.

Investigation of the effects of heat shock and agents which induce a heat shock response on the induction of differentiation of HL-60 cells.

A heat shock of 42.5-43.5 degrees C for 1 h applied to HL-60 promyelocytic leukemia cells induced the appearance of between 13 and 34% (n = 6) of cells which showed characteristics of mature metamyelocytes/granulocytes. This is the first time a physical agent has been shown to induce the differentiation of this leukemic cell line. The treatment of HL-60 cells with a variety of agents which have been documented to stress cells and induce thermotolerance or a heat shock-like response also induced granulocyte-like differentiation: continuous treatment for 4 days with ethanol (213 mM), sodium arsenite (6 microM), cadmium sulfate (60 microM), lidocaine (3 mM), and procaine (5 mM) induced 73, 54, 14, 54, and 55% of cells, respectively, to reduce the dye nitro blue tetrazolium. They were also capable of the phagocytosis of yeast particles. Examination of differentiated cells showed that those treated with ethanol, arsenite, lidocaine, and procaine also expressed nonspecific esterase activity, typical of monocytes, but did not adhere to plastic and had a cellular and nuclear morphology consistent with differentiation to metamyelocytes. Analysis of protein synthesis of HL-60 cells treated with 170 mM N-methylformamide, by the pulse labeling of cells for 2 h with [14C]leucine at various times, showed that the constitutive synthesis of both the Mr 90,000 and 70,000 heat shock proteins fell substantially after 2 h of exposure to N-methylformamide. When HL-60 cells were incubated with 1 M N-methylformamide, a toxic concentration of this agent, or were heat shocked, the synthesis of both the Mr 70,000 and Mr 90,000 proteins was induced. We propose that changes in heat shock protein synthesis may be an important element of the induction of differentiation of HL-60 cells, particularly as these proteins have recently been shown to regulate the stability of oncogene proteins, such as myc (Lüscher, B., and Eisenman, R. N., Mol. Cell Biol., 8: 2504-2512, 1988).

Elevation of leukemic cell intracellular calcium by the ether lipid SRI 62-834.

SRI 62-834 is a novel antineoplastic ether lipid which is currently undergoing a Phase 1 clinical trial in the United Kingdom. Its mechanism of action has not been defined. Incubation of 7.5 X 10(6)/ml HL-60 human myelomonocytic leukemia cells with between 10 and 50 microM SRI 62-834 brought about a concentration-dependent, biphasic rise in intracellular calcium, as measured by the calcium-sensitive fluorescent dye Quin-2 AM. Incubation with 30 microM SRI 62-834 elevated intracellular calcium from 110 to 415 nM after 10 min in a typical experiment; this concentration inhibited cell growth by greater than 90% (50% inhibition of growth was observed at 8 microM). The calcium channel blockers verapamil and prenylamine did not inhibit the SRI 62-834-induced elevation of intracellular calcium. Incubation of SRI 62-834 with K562 erythroblastic leukemia cells also brought about a rise in the intracellular calcium. The growth of K562 cells was less sensitive to SRI 62-834 (dose to produce 50% growth inhibition, 65 microM) compared to HL-60 cells, and significant intracellular calcium rises, which were monophasic, required greater than 40 microM SRI 62-834. At a concentration of SRI 62-834 which inhibited both HL-60 and K562 growth by 90% (30 and 140 microM, respectively) an equivalent rise in intracellular calcium was observed (circa 400 nM). Preincubation of HL-60 or K562 cells with 1 to 100 nM 12-O-tetradecanoylphorbol-13-acetate for 10 min prior to the addition of SRI 62-834 inhibited the rise in intracellular calcium in a concentration-dependent manner. It is suggested that SRI 62-834-induced changes in intracellular calcium may contribute to its cytotoxicity and that the rise is not due to an early and grossly disruptive effect of the agent on membrane structure.

Effects of N-methylformamide on the growth, cell cycle, and glutathione status of murine TLX5 lymphoma cells.

The growth of the murine TLX5 lymphoma is inhibited in vivo by administration of N-methylformamide (Gescher, A., et al., Br. J. Cancer, 45: 843-850, 1982). Continuous incubation of TLX5 murine lymphoma cells in vitro with N-methylformamide for 72 h, at concentrations of between 43 and 170 mM (0.25 and 1% v/v), brought about a concentration-dependent decrease in growth rate (50% inhibitory concentration = 68 mM) and viability. Cell replication was decreased by 37% after 48 h exposure to 106 mM N-methylformamide, while viability was maintained at 82%. Analysis of the distribution of these cells in the cell cycle by flow cytofluorimetry showed a 23% increase in the proportion of G1 cells and a fall in the proportion of cells in the S and G2/M phases. As the drug concentration and time of exposure to N-methylformamide were increased, with an associated reduction in cell replication and viability, the proportion of G1 cells rose. When TLX5 cells were washed free of N-methylformamide after an exposure to 106 mM for 48 h and cultured in drug-free medium, the cells returned to exponential growth and to a normal cell cycle distribution. Clonogenic assays showed that the recovery of proliferation, after removal of the drug, was due to that of all those cells which, in a parallel experiment, excluded the dye trypan blue. It is concluded that the cessation of replication and the accumulation of cells in G1 of the cell cycle, after treatment with N-methylformamide, are probably not events representative of terminal differentiation but rather of cytostasis, which was accompanied by rapid cell death. Coincident with the reduction of TLX5 cell proliferation caused by N-methylformamide and the accumulation of cells in G1, cellular glutathione concentrations fell by 80%. A similar fall was induced by treatment of the cells with D,L-buthionine[S,R]-sulfoximine (5 microM) for 48 h, but this treatment had no effect on cell growth.

Effects of the antitumor agent 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4(3 H)-one on the DNA of mouse L1210 cells.

L1210 murine leukemia cells were treated in vitro with the novel antineoplastic agent 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d] -1,2,3,5-tetrazin-4(3H)-one (M&B 39565), and its interaction with cellular DNA was assessed by alkaline elution. DNA interstrand cross-link and DNA-protein cross-link formation was quantified with regard to drug concentration and length of incubation time after a 2-hr incubation period with drug. Cytotoxicity, as measured by colony formation assays, and DNA damage caused by M&B 39565 were compared with those caused by a breakdown product of M&B 39565, 5-[3-(2-chloroethyl)triazen -1-yl]imidazole-4-carboxamide (MCTIC) and also with 1-(2-chloroethyl)-1-nitrosourea (CNU). Both MCTIC and CNU decompose to yield a 2-chloroethyldiazo species which is capable of alkylating DNA. At equimolar concentrations, all three drugs possessed similar in vitro cytotoxicities; at equitoxic concentrations, they produced similar levels of DNA interstrand cross-linking. The time course for cross-link formation was different for CNU when compared with MCTIC and M&B 39565, with peaks at 6 hr (CNU) and 9 hr (M&B 39565 and MCTIC). This study suggests that M&B 39565 is cytotoxic against L1210 leukemia cells as a consequence of DNA interstrand cross-link formation, probably via its breakdown product MCTIC.

DNA cross-linking and cytotoxicity in normal and transformed human cells treated in vitro with 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d] -1,2,3,5-tetrazin-4(3H)-one.

Normal (IMR-90) and SV40-transformed (VA-13) human embryo cells were treated with 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d] -1,2,3,5-tetrazin-4(3H)-one (M&B 39565), and the effects of the drug on cell viability and cellular DNA integrity were studied. The effects of M&B 39565 were compared with one of its potential decomposition products 5-[3-(2-chloroethyl)triazen-1 -yl]imidazole-4-carboxamide (MCTIC). M&B 39565 and MCTIC were 5- to 6-fold more toxic to VA-13 cells than to IMR-90 cells for drug concentrations which produced a 2-log cell kill, as measured by colony-forming assays. Using alkaline elution analysis, VA-13 cells exhibited concentration-dependent DNA interstrand cross-link formation. In IMR-90 cells, little or no interstrand cross-link formation was detected. The DNA interstrand cross-link formation in VA-13 cells was found to peak 12 hr after drug removal. A linear correlation between DNA interstrand cross-link formation and log cell kill was observed in VA-13 cells but not in IMR-90 cells. DNA-protein cross-link formation was found to be comparable in both cell lines for each drug, suggesting that drug penetration and intracellular drug reactivity were similar. Initial chemical decomposition studies suggest that both M&B 39565 and MCTIC may produce a chloroethyldiazo species. This species has been implicated in the formation of chloroethyl-DNA adducts which convert to DNA interstrand cross-links in mammalian cells treated with chloroethylnitrosoureas [Erickson et al., Nature (Lond.), 288: 727, 1980]. These data suggest that DNA interstrand cross-link formation may be a common mechanism for the in vitro cytotoxicity of M&B 39565 and MCTIC.

Germinal center-derived signals act with Bcl-2 to decrease apoptosis and increase clonogenicity of drug-treated human B lymphoma cells.

Bcl-2 suppresses drug-induced apoptosis in vitro, although in many cases, this results only in a delayed onset of cell death. In vivo survival signals from the extracellular environment may also contribute to drug resistance and may act with Bcl-2 to promote long-term cell survival. Ligation of CD40 on B-lymphocytes in germinal centers (GCs) can suppress apoptosis induced by calcium ionophore or anti-IgM in vitro. We asked whether a combination of Bcl-2 expression and the provision of a culture environment that mimicked that of the GC [CD40 ligation and interleukin 4 (IL-4)] could increase the ability of B lymphoma cells to resist drug-induced apoptosis. A Burkitt lymphoma (BL) cell line transfected with either human bcl-2 (BL-bcl-2) or control plasmid (BL-Sv2) was used to examine the effects of Bcl-2 overexpression on the cellular response and long-term survival after treatment with the DNA-alkylating drug chlorambucil (CMB) in the presence or absence of CD40 ligation and IL-4. Administration of 20 microM CMB completely prevented cell proliferation. This was associated with an increase in p53 protein levels within 24 h, without an elevation in p21, Bax, or Mdm2 proteins. Analyses of cell cycle distribution and of cyclin B expression demonstrated that both cell lines arrested at G2/M, where they died. Fifty % of BL-Sv2 cells died within 2 days, whereas 50% cell death was not observed in the BL-bcl-2 cultures until 6 days had passed. Cross-linking of CD40 with a monoclonal antibody elevated Bcl-xL protein levels by 3 h and also provided a delay in CMB-induced death. Ninety-six h after the addition of 20 microM CMB, 78% of the BL-Sv2 cells were apoptotic, whereas ligation of CD40 on BL-Sv2 cells reduced the proportion of apoptotic cells to 38%. Overexpression of Bcl-2 (in BL-bcl-2 cells) reduced apoptosis to 41%. However, when the BL-bcl-2 cells were treated with CMB together with ligation of CD40, apoptosis was reduced further to only 17% at 96 h. The Bcl-2-mediated delay in the execution of CMB-induced apoptosis did not translate significantly to increased clonogenicity. In contrast, the provision of BL-Sv2 cells with an ability to interact with the adhesion molecule vascular cell adhesion molecule-1, CD40 ligation, and IL-4 significantly increased clonogenic survival, and this was improved in BL-bcl-2 cells exposed to these GC-derived signals. These data demonstrate that the kinetics of drug-induced apoptosis can be modulated by Bcl-2 as well as by IL-4, vascular cell adhesion molecule-1, and CD40 ligation, the latter possibly involving the function of Bcl-xL. That these factors appear to act together to enhance proliferative potential after DNA damage has important implications regarding the development of drug resistance in B-cell lymphomas and future strategies for improved chemotherapy.