Implications of the involvement of the endoplasmic reticulum stress pathway in drug-induced apoptosis.

Apoptosis occurs by distinct pathways that involve the cell surface, mitochondria or the endoplasmic reticulum. Previous studies had shown that deoxyadenosine-resistant L1210 cells (Y8) proceeded to apoptosis under conditions in which the parental L1210 cell line (WT) did not undergo an apoptotic response. Combinations of drugs, acting at different molecular targets, markedly potentiated the apoptotic response in the Y8 cells without inducing apoptosis in the WT cells. In the present study, induction of apoptosis by parthenolide and BAY 11-7085, drugs that targeted nuclear factor kappa B activation, was blocked by the presence of N-acetylcysteine (NAC). On the other hand, the levels of apoptosis induced by parthenolide or BAY 11-7085 were increased by pre-treatment of the cells with glutathione lowering L-buthionine-(S,R)-sulfoximine (BSO). Western blot analyses showed that the levels of the stress proteins, Grp 78 and Gadd 153 were reduced in the parthenolide-treated Y8 cells, but not in those co-treated with NAC. Protection of the cells from apoptosis induced by parthenolide or BAY 11-7085 by NAC was relatively specific as the induction of apoptosis in the Y8 cells by MG-132, flavopiridol, Gemcitabine or PRIMA-1 was not decreased by NAC. These data suggest that multiple pathways, one of which is ER-stress induced, may ultimately be involved and interactive in the induction of apoptosis in specific cell lines.

Understanding interactions between and among apoptosis inducing pathways in tumor cells.

It has become increasingly clear that the induction of apoptosis in tumor cells can occur by at least three different pathways involving the cell surface receptors, the mitochondria and the endoplasmic reticulum. Specific drugs and conditions can trigger the apoptotic response via one of the three known pathways. What is less clear is how these three pathways can interact synergistically or antagonistically to influence a common convergence step leading to the programmed cell death. In this report we present data to show that fenretinide (a synthetic retinoid) potentiates the apoptotic effects of parthenolide (a drug that inhibits the activation of NF-kappa B) and BAY 11-7085 (an inhibitor of I-kappa B-alpha kinase). This potentiation of apoptosis by fenretinide is seen in the p53-deficient, deoxyadenosine-resistant L1210 cells, but not in the parental L1210 cells that express a mutant p53. These effects are seen at a concentration of fenretinide that have little effect by itself. These data strongly suggest that fenretinide activates or inhibits some step or pathway that interacts with the inhibition of NF-kappa B activation required for the apoptotic response. Since parthenolide, BAY 11-7085 and fenretinide are well known drugs in clinical trials, an understanding of the nature of the interactions between or among the apoptotic pathways could lead to the design of better clinical protocols using these drugs that will promote apoptosis in tumor cells.

Bis(2-hydroxybenzylidene)acetone, a potent inducer of the phase 2 response, causes apoptosis in mouse leukemia cells through a p53-independent, caspase-mediated pathway.

Bis(2-hydroxybenzylidene)acetone is a potent inducer of the phase 2 response through the Keap1-Nrf2-ARE pathway. This double Michael reaction acceptor reacts directly with Keap1, the sensor protein for inducers, leading to enhanced transcription of phase 2 genes and protection against oxidant and electrophile toxicities. In our efforts to identify potent chemoprotective agents, we found that in rapidly growing murine leukemia cells (L1210) low concentrations (in the submicromolar range) of bis(2-hydroxybenzylidene)acetone markedly increased the activities of NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) and glutathione reductase, and the levels of total glutathione, three markers of the phase 2 response. In contrast, at high concentrations (in the micromolar range) the same compound caused G2/M cell cycle arrest and apoptosis. Importantly, a mutant L1210 cell line (Y8), selected for resistance to deoxyadenosine and lacking expression of p53 protein, was considerably more sensitive to the apoptotic effects of bis(2-hydroxybenzylidene)acetone. When caspase activities were evaluated in cell-free extracts prepared from treated wild type or mutant L1210 cells, the activities of caspase-3, the terminal caspase in the cascade leading to apoptosis, and caspase-10 were found to be markedly elevated. The activities of other caspases measured, caspase-1, -6 and -8, were not appreciably affected. Thus, both induction of the phase 2 response and p53-independent, caspase-3-mediated apoptosis could act cooperatively in chemoprotection. The concentration-dependent differential effects on these two pathways should be carefully considered in mechanistic explanations and strategic designs.

Critical roles of glutamine as nitrogen donors in purine and pyrimidine nucleotide synthesis: asparaginase treatment in childhood acute lymphoblastic leukemia.

Asparaginase is a key component of the chemotherapy protocols used in the treatment of acute lymphoblastic leukemia (ALL). The current treatment protocols are remarkable in that childhood ALL cure rates are approaching 85%. As the name implies, asparaginase catalyzes the deamination of asparagine to aspartic acid. What is not generally realized is that asparaginase also catalyzes, essentially to the same extent, the removal of the amide nitrogen from glutamine to form glutamic acid. Glutamine is a required substrate for three enzymes involved in the de novo synthesis of purine nucleotides and two enzymes involved in the de novo synthesis of pyrimidine nucleotides. In this review, the specific roles of glutamine in the de novo synthesis of nucleotides are defined and an appropriate explanation for the cell cycle arrest and cytotoxicity induced in proliferating malignant lymphoblasts by asparaginase treatment is provided.

Effects of PRIMA-1 on wild-type L1210 cells expressing mutant p53 and drug-resistant L1210 cells lacking expression of p53: necrosis vs. apoptosis.

The effects of PRIMA-1 on wild-type (WT) mouse leukemia L1210 cells and drug-resistant L1210 cells (Y8) were studied with respect to the induction of apoptosis and necrosis in these cell lines. The WT L1210 cells express mutant p53 while the Y8 L1210 cells do not express p53 mRNA or protein, but do express WAF1/p21 and Gadd 45 mRNA's and proteins. It was found that, in response to treatment with PRIMA-1, the WT L1210 cells became necrotic with little apoptosis while the Y8 L1210 cells showed a much higher level of apoptosis than necrosis. Flavopiridol in combination with PRIMA-1 caused a synergistic increase in necrosis in the WT L1210 cells while LY 294002 in combination with PRIMA-1 caused a synergistic increase in apoptosis in the Y8 L1210 cells. These studies showed that PRIMA-1 had an effect not only on cells expressing mutant p53, but also on cells that do not express p53, suggesting that PRIMA-1 and PRIMA-1-like molecules have multiple sites of action independent of restoring p53 function and that these can interact with other signaling pathways involving CDK's and PI3 kinases.

Induction of apoptosis in p53-deficient L1210 cells by an I-kappa-B-alpha-inhibitor (Bay 11-7085) via a NF-kappa-B-independent mechanism.

The effects of Bay 11-7085, an inhibitor of I-kappa-B-alpha kinase, were compared in the wild-type and deoxyadenosine-resistant mouse leukemia cell lines. At higher concentrations, Bay 11-7085 caused apoptosis and necrosis in the two cell lines. However, at much lower concentrations of Bay 11-7085, the deoxyadenosine-resistant cells became much more apoptotic than the parental wild-type L1210 cells. Under these conditions (low drug concentrations), the level of apoptotic cells far exceeded the fraction of necrotic cells. The apoptotic effects of Bay 11-7085 on the deoxyadenosine-resistant cells was both time- and concentration-dependent. Caspase-3 activity was activated in the Bay 11-7085-treated cells. The molecular basis for the difference in the apoptotic response between the wild-type and deoxyadenosine-resistant L1210 cells is not defined at this time, but these cell lines may provide a comparative model system in which differences in the cells that lead to apoptotic or necrotic responses to drugs can be defined and used in development of appropriate drugs for clinical use.

Blockage of cyclin cdk's, PKC and phosphoinositol 3-kinase pathways leads to augmentation of apoptosis in drug-resistant leukemia cells: evidence for interactive effects of flavopiridol, LY 294002, roscovitine,wortmannin and UCN-01.

A mouse leukemia L1210 cell line (Y8), selected for resistance to deoxyadenosine, has a markedly altered phenotypic expression that includes loss of sensitivity to dATP as an allosteric inhibitor of ribonucleotide reductase, increased expression of c-myc, c-fos and WAF1/p21, but decreased expression of p53. In addition, the Y8 cells have a Very strong apoptotic response to a variety of agents under conditions in which the parental wild-type cells do not apoptose. In these studies, we show that flavopiridol (a cdk inhibitor) causes the Y8 cells to undergo apoptosis via a caspase-3 activation process. The apoptotic response to flavopiridol is markedly enhanced by LY294002. Data also show that the apoptotic response of the Y8 cells to roscovitine (a cdk inhibitor) is enhanced by UCN-01 (a PKC inhibitor). These data show that simultaneous blockage of specific pathways leads to increased apoptosis in the Y8 cells with essentially no effects on the parental wild-type L1210 cells.

Phenolic compounds, sodium salicylate and related compounds, as inhibitors of tumor cell growth and inducers of apoptosis in mouse leukemia L1210 cells.

The effects of a series of phenolic compounds were compared to the effects of sodium salicylate (2-hydroxybenzoate) on the growth, cell cycle and apoptotic effects in wild-type (WT) and deoxyadenosine-resistant (Y8) L1210 leukemia cells. These compounds included: salicylaldehyde, salicylaldoxime, salicylhydroxamic acid, salicylamide, 5-aminosalicylate and 5-sulfosalicylate. The IC50 values for inhibition of tumor cell growth ranged from 40 microM for salicylaldhyde to greater than 4 mM for 5-sulfosalicylate. There appeared to be an excellent correlation between the IC50 value for a compound and the ratio of octanol/aqueous distribution. Salicylamide caused a G2/M block in both the WT and Y8 L1210 cells, while salicylalehyde caused a G0/G1 block in both the WT and Y8 cells. Salicylamide and salicylaldoxime caused a much greater apoptotic effect in the Y8 cells than in the parental WT L210 cells. These data suggest that salicylaldehyde and salicylaldoxime, the most active compounds in this series, may provide the lead chemicals from which other more active drugs can be synthesized.

In vivo growth of mouse leukemia L1210 cells with metabolic alterations in the subunits of ribonucleotide reductase.

Mouse leukemia L1210 cells selected for resistance to drugs targeted specifically at each of the protein subunits of ribonucleotide reductase were studied for their ability to grow in vivo. The life-span of the mice injected with hydroxyurea-resistant L1210 cells, which have elevated levels of the mRNA and protein for the non-heme iron (NHI, R2) subunit of ribonucleotide reductase, was approximately twice that of the mice injected with equal numbers of the parental wild-type L1210 leukemia cells. The life-span of mice injected with the L1210 cells that had alterations in the effector-binding subunit (EB, R1) was considerably shorter than the mice injected with the parental wild-type L1210 cells. These results provide direct evidence that tumor cells with alterations in the properties of ribonucleotide reductase grow differently in vivo, with defined effects on the host mouse that cause either an increased survival time or a decreased survival time compared to the effects of wild-type L1210 leukemia cells on tumor-bearing mice.

Lipophilic derivatives of cyclam as new inhibitors of tumor cell growth.

Two new lipophilic tetraazamacrocycles were prepared and, in contrast to non-lipophilic analogs, found to be potent inhibitors of tumor cell growth in vitro with IC50 values below 10 micromolar.

Lactacystin, a proteasome inhibitor, potentiates the apoptotic effect of parthenolide, an inhibitor of NFkappaB activation, on drug-resistant mouse leukemia L1210 cells.

An L1210 cell line (Y8) selected for resistance to deoxyadenosine does not express p53 mRNA or protein but expresses WAF1/p21 even under basal conditions. The Y8 cell line had been previously shown to have an increased apoptotic response to a variety of agents that included DNA damaging agents, kinase inhibitors and drugs directed at NFkappa B activation. In this study we show that lactacystin (LC, an inhibitor of proteasome activity) in combination with parthenolide (PA) caused a synergistic increase in the apoptotic fraction of the Y8 cells. LC (2.5 microM) alone and PA (5.0 microM) caused less than 20% of the Y8 cells to undergo apoptosis. However, the combination of LC (2.5 microM) plus PA (5.0 microM) caused 60% of the Y8 cells to undergo apoptosis. The combination of drugs had no effects on the parental wild-type L1210 cells. Pretreatment of the intact Y8 cells with the caspase-3 inhibitor, Ac-DEVD-CHO, resulted in a marked decrease in the apoptosis caused by the LC plus PA combination. Cell-free extracts prepared from the LC plus PA combination-treated cells had activated caspase activities in the caspase cascade: caspase-3 >> caspase-8 > caspase-6 and caspase-10. These results suggest that there are interacting pathways involving aspects of NFkappa B activation and proteasome activity that could be exploited in therapy directed at p53-deficient tumor cells that would lead to caspase-3 activation and apoptosis bypassing the p53-dependent chemotherapy insensitivity.