'Seed' analysis of off-target siRNAs reveals an essential role of Mcl-1 in resistance to the small-molecule Bcl-2/Bcl-XL inhibitor ABT-737.

ABT-737 is a subnanomolar inhibitor of the antiapoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w. Although ABT-737 triggers extensive cell death in many small-cell lung carcinoma (SCLC) cell lines, some of the SCLC cell lines and the majority of the cancer cell lines derived from other solid tumors were found to be resistant to ABT-737. To better understand the mechanism of resistance to ABT-737, we screened a short interfering RNA library consisting of short interfering RNA against 4000 'druggable' targets in an SCLC-derived cell line, NCI-H196. By comparing the knockdowns with phenotypes, all of the three top 'hits' from the screen were found to result from off-target gene silencing. Interestingly, the three off-target siRNAs were found to knock down an antiapoptotic Bcl-2 family protein Mcl-1 owing to the complementation between their seed regions with the 3' untranslated region (3' UTR) of Mcl-1. Furthermore, reducing the level of Mcl-1 using siRNAs or the small-molecule compounds Bay43-9006 and Seliciclib was sufficient to overcome the resistance to ABT-737 in the resistant SCLC cell line and cancer cell lines derived from other solid tumors. These results provide further evidence that Mcl-1 is the major factor that causes resistance to ABT-737 in cancer cells derived from diverse solid tumors, and the combination of Mcl-1 downregulating agents with ABT-737 could be potent therapeutic regimens for patient with ABT-737-resistant SCLC and many other types of solid tumors.

Evaluation of positive controls for the in vitro unscheduled DNA synthesis assay using hepatocytes from induced (Aroclor 1254) and uninduced male cynomolgus monkey.

We have evaluated the use of four different positive control compounds for assessing UDS in monkey hepatocytes and have found three of these, methylmethanesulfonate, benzo[a]pyrene, and dimethylbenz[a]anthracene, to produce strong positive responses in vitro. Dimethylnitrosamine induced only weak responses. We also report that the strength of the response induced by procarcinogens was not enhanced in hepatocytes taken from Aroclor 1254-pretreated monkeys, even though substantial induction of cytochrome P450 enzymes was demonstrated in these cells. These studies raise the question of the utility of employing an in vivo induction system to enhance the monkey UDS assay.

Differential toxicity of an inhibitor of mitochondrial respiration in canine hepatocytes and adrenocortical cell cultures.

PD132301-2, a novel inhibitor of acyl-CoA: cholesterol acyltransferase, was previously shown to be an inhibitor of mitochondrial respiration and an adrenal toxicant in several species. To investigate potential mechanisms of tissue-specific toxicity in vivo, dog adrenocortical and hepatocyte cell cultures were exposed to 0.01-30 mum PD132301-2 for 0-24 hr. Cell viability was assessed by neutral red uptake or release assays. Cytotoxicity was observed in adrenocortical cells at 0.01 mum or above after 2hr of exposure, while 30 mum was not toxic to hepatocytes after 24 hr of exposure. Decreases in adrenocortical cell viability were attenuated in the presence of 20 mm fructose, a glycolytic substrate, and fructose protection was in turn blocked by the glycolytic inhibitor NaF (1 mm). In contrast, PD132301-2-induced hepatocellular toxicity was evident only following pretreatment with NaF or metyrapone, a broad-spectrum cytochrome P-450 inhibitor. With either co-treatment, hepatocyte viability was reduced 50% after 6 hr at 1 mum or more PD132301-2. These data indicate that the relative sensitivity of adrenocortical cells to the cytotoxic effects of PD132301-2 may be due, in part, to their relative lack of the metabolic detoxification and glycolytic reserve capacities that appear to protect hepatocytes from PD132301-2 toxicity.

Biochemical alterations in guinea pig adrenal cortex following administration of PD 132301-2, an inhibitor of acyl-CoA:cholesterol acyltransferase.

To assess whether previously reported ultrastructural alterations of adrenocortical mitochondria induced by the acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor PD 132301-2 are accompanied by functional deficits in tissue energy stores, phosphorylated adenine nucleotide levels in guinea pig adrenal cortex were quantitated. Adrenals of male guinea pigs were obtained at 1, 2, 6, or 24 hours after oral administration of 100 mg/kg PD 132301-2 or 0.5% methylcellulose vehicle. In treated animals, ATP levels and ATP/ADP ratios were decreased approximately 50% with concurrent increases in AMP. Calculated energy charge was also decreased; these decreases were maximal by 6 hours. Cholesterol esterification in adrenal cortex was inhibited, resulting in progressive accumulation of free cholesterol up to 3-fold over control by 24 hours, consistent with the ACAT inhibitory activity of the drug. These data suggest that PD 132301-2 distributes to the adrenal cortex where early alterations of tissue bioenergetics occur in a time frame consistent with ultrastructural alterations of mitochondria.

ATP depletion is associated with cytotoxicity of a novel lipid regulator in guinea pig adrenocortical cells.

A novel lipid regulator (PD132301-2) produces degeneration and necrosis of adrenal fasciculata in guinea pigs. Primary adrenocortical cell cultures from male Hartley guinea pigs were utilized to investigate potential mechanisms of this toxicity. Concentration-dependent loss of viability, measured by neutral red (NR) accumulation or MTT reduction, was observed within 6 hr at concentrations of 0.01 to 10 microM PD132301-2. At 10 microM, NR and MTT indices were 50% of those of control after 6 hr exposure. Maximal decreases in NR and MTT indices to 20% of control values occurred by 24 hr at > or = 1 microM PD132301-2. Adenine nucleotide analysis after PD132301-2 challenge indicated that ATP depletion preceded loss of viability. At 10 microM PD132301-2, ATP levels were 80% of those of control after 30 min and 25% of those of control after 6 hr. Supplementation of glucose-free buffer with 20 mM fructose protected adrenocortical cells from PD132301-2-induced toxicity. Fructose protection was blocked by inhibiting glycolysis with 1 mM sodium fluoride. Pretreatment of cultures with 100 microM metyrapone, an inhibitor of cytochrome P-450, did not block cytotoxicity induced by 10 microM PD132301-2, but did block cytotoxicity of 100 microM o,p'-DDD. In adrenocortical mitochondrial preparations, inhibition of respiration by PD132301-2 was site II-specific. Both state 3 and state 4 respiration were inhibited 50-75% at 1-30 microM PD132301-2. Thus, ATP depletion resulting from direct inhibition of mitochondrial respiration is a critical early event in adrenocortical cytotoxicity of PD132301-2.

Selective alteration of cytokeratin intermediate filament by cyclosporine A is a lethal toxicity in PTK2 cell cultures.

The cytoplasm of eukaryotic cells contain a series of three filamentous structures, microtubules, microfilaments, and intermediate filaments that are termed the cytoskeleton. Cytokeratin, one type of intermediate filament, has no known physiological function, yet, can comprise up to 30% of the total cytoplasmic protein content. As there are no selective toxins to cytokeratins, it is not known if alterations to these hydrophobic filaments is a lethal event. Cyclosporine A, a novel hydrophobic immunosuppressant compound used to prevent allograft rejection, may show a selective toxicity to the cytokeratin filaments. This effect is seen in PtK2 cell cultures as a single large perinuclear aggregate of collapsed cytokeratin filaments (5 mM, 72 hr). Microtubules and microfilaments are not affected in PtK2 cell cultures (5 mM, 72 hr). Increased LDH levels into cell culturing media occur soon after cyclosporine exposure to PtK2 cell cultures (5 mM, 2 hr). Cytokeratin filaments show no changes at 12 hr exposure but show thickening, decreased plasma membrane attachments and some peri-nuclear ring formations at 24 hr (5 mM, 24 hr). Cyclosporine G, an analog of cyclosporine A, does not exhibit the cytokeratin filament collapse (5 mM, 72 hr). The effect of cyclosporine A on DNA binding protein (Mr 64 kd), believed to be a nuclear scaffolding protein related to intermediate filaments, exhibited an early invagination and folding of the nuclear membrane (5 mM, 4 hr). Due to a hydrophobic bonding potential between cyclosporine A and cytokeratin and cytokeratin-like intermediate filaments, cyclosporin A may be a selective cytokeratin toxin. Alteration of the cytokeratin filaments in PtK2 cell cultures may be a lethal event.