Mitochondriotoxic compounds for cancer therapy.

One of the hallmarks of cancer cells is their increased resistance to apoptosis induction. Alterations in many apoptosis regulators belonging to the intrinsic pathway confer emerging neoplastic cells with a selective growth advantage in the hostile tumor microenvironment. The realization that those same defects contribute to resistance to radiation and chemotherapeutic agents have prompted the unrelenting search for mitochondria-targeted compounds for the treatment of cancer. Mitochondria play a central role in the process of cell death. They serve as integrators of upstream effector mechanisms. Most importantly, mitochondrial outer membrane permeabilization becomes a commitment point during cell death. Thus, strategies aimed at directly triggering this event by either blocking the activity of antiapoptotic factors or by interfering with vital mitochondrial functions may help to overcome resistance to standard cancer therapy.

Cloning, tissue expression, and chromosomal location of the mouse insulin receptor substrate 4 gene.

The insulin receptor substrates (IRSs) are key proteins in signal transduction from the insulin receptor. Recently, we discovered a fourth member of this family, designated IRS-4, cloned its complementary DNA from the human embryonic kidney 293 cell line, and characterized its signaling properties in this cell line. As part of an investigation of the physiological role of this IRS, we have now cloned the mouse IRS-4 gene and determined its tissue expression and chromosomal location. The coding region of the mouse IRS-4 gene contains no introns, and in this regard is the same as that of the genes for IRS-1 and -2. The predicted amino acid sequence of mouse IRS-4 is highly homologous with that of human IRS-4; the pleckstrin homology domain, the phosphotyrosine-binding domain, and the tyrosine phosphorylation motifs are especially well conserved. The tissue distribution of IRS-4 in the mouse was determined by analysis for the expression of its messenger RNA by RT-PCR and for the protein itself by immunoprecipitation and immunoblotting. The messenger RNA was detected in skeletal muscle, brain, heart, kidney, and liver, but the protein itself was not detected in any tissue. These results indicate that IRS-4 is a very rare protein. The chromosomal locations of the mouse IRS-4 and IRS-3 genes were determined by interspecific back-cross analysis and were found to be on chromosomes X and 5, respectively. As the mouse genes for IRS-1 and -2 are on chromosomes 1 and 8, respectively, each IRS gene resides on a different chromosome.

Cancer-associated IDH mutations: biomarker and therapeutic opportunities.

The discovery of somatic mutations in the isocitrate dehydrogenase (IDH) enzymes through a genome-wide mutational analysis in glioblastoma represents a milestone event in cancer biology. The nature of the heterozygous, point mutations mapping to arginine residues involved in the substrate binding inspired several research teams to investigate their impact on the biochemical activity of these enzymes. Soon, it became clear that the mutations identified impaired the ability of IDH1 and IDH2 to catalyze the conversion of isocitrate to α-ketoglutarate (αKG), whereas conferring a gain of a novel enzymatic activity leading to the reduction of αKG to the metabolite D2-hydroxyglutarate (D-2HG). Across glioma as well as several hematologic malignancies, mutations in IDH1 and IDH2 have shown prognostic value. Several hypotheses implicating the elevated levels of D-2HG and tumorigenesis, and the therapeutic potential of targeting mutant IDH enzymes will be discussed.

Mice lacking insulin receptor substrate 4 exhibit mild defects in growth, reproduction, and glucose homeostasis.

The insulin receptor substrates (IRSs) function in insulin signaling. Four members of the family, IRS-1 through IRS-4, are known. Previously, mice with targeted disruption of the genes for IRS-1, -2, and -3 have been characterized. To examine the physiological role of IRS-4, we have generated and characterized mice lacking IRS-4. Male IRS-4-null mice were approximately 10% smaller in size than wild-type male mice at 9 wk of age and beyond, whereas the female null mice were of normal size. Breeding pairs of IRS-4-null mice reproduced less well than wild-type mice. IRS-4-null mice exhibited slightly lower blood glucose concentration than the wild-type mice in both the fasted and fed states, but the plasma insulin concentrations of the IRS-4-null mice in the fasted and fed states were normal. IRS-4-null mice also showed a slightly impaired response in the oral glucose tolerance test. Thus the absence of IRS-4 caused mild defects in growth, reproduction, and glucose homeostasis.

Insulin receptor substrate 4 supports insulin- and interleukin 4-stimulated proliferation of hematopoietic cells.

Signaling from the activated insulin receptor is initiated by its tyrosine phosphorylation of the insulin receptor substrates (IRSs). The IRSs then act as docking/effector proteins for various signaling proteins containing src homology 2 domains. Four members of the IRS family, designated IRS-1 through IRS-4, have been identified. Although these IRSs show considerable structural homology, the extent to which they overlap in functions has not been explored in detail. The 32D hematopoietic cell line, which contains no detectable amounts of any IRS, provides a system in which to determine whether an IRS supports cell proliferation. Previous studies have shown that introduction of IRS-1 or -2 into 32D cells overexpressing the insulin and IL-4 receptors (32D-R cells) enables the cells to undergo mitogenesis in response to insulin and IL-4. In the present study, we have examined IRS-4, a member of the IRS family that we recently discovered, in this system. Expression of IRS-4 in 32D-R cells permitted the cells to undergo mitogenesis and continuous proliferation in response to insulin and IL-4. Immunoblotting of phosphotyrosine proteins showed that insulin and IL-4 elicited the tyrosine phosphorylation of IRS-4 in these cells. Thus, IRS-4, like IRS-1 and -2, can function in the signal transduction pathways linking insulin and IL-4 receptors to cell proliferation.

A novel 160-kDa phosphotyrosine protein in insulin-treated embryonic kidney cells is a new member of the insulin receptor substrate family.

We have previously identified a 160-kDa protein in human embryonic kidney (HEK) 293 cells that undergoes rapid tyrosine phosphorylation in response to insulin (PY160) (Kuhné, M. R., Zhao, Z., and Lienhard, G. E. (1995) Biochem. Biophys. Res. Commun. 211, 190-197). The phosphotyrosine form of PY160 was purified from insulin-treated HEK 293 cells by anti-phosphotyrosine immunoaffinity chromatography, the sequences of peptides determined, and its cDNA cloned. The PY160 cDNA encodes a 1257-amino acid protein that contains, in order from its N terminus, a pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain, and, spread over the C-terminal portion, 12 potential tyrosine phosphorylation sites. Several of these sites are in motifs expected to bind specific SH2 domain-containing proteins: YXXM (7 sites), phosphatidylinositol 3-kinase; YVNM (1 site), Grb-2; and YIEV (1 site), either the protein-tyrosine phosphatase SHP-2 or phospholipase Cgamma. Furthermore, the PH and PTB domains are highly homologous (at least 40% identical) to those found in insulin receptor substrates 1, 2, and 3 (IRS-1, IRS-2, and IRS-3). Thus, PY160 is a new member of the IRS family, which we have designated IRS-4.

Characterization of insulin receptor substrate 4 in human embryonic kidney 293 cells.

We recently cloned IRS-4, a new member of the insulin receptor substrate (IRS) family. In this study we have characterized IRS-4 in human embryonic kidney 293 cells, where it was originally discovered. IRS-4 was the predominant insulin-elicited phosphotyrosine protein in these cells. Subcellular fractionation revealed that about 50% of IRS-4 was located in cellular membranes, and immunofluorescence indicated that IRS-4 was concentrated at the plasma membrane. Immunoelectron microscopy conclusively established that a large portion of the IRS-4 was located at the cytoplasmic surface of the plasma membrane in both the unstimulated and insulin-treated states. IRS-4 was found to be associated with two src homology 2 (SH2) domain-containing proteins, phosphatidylinositol 3-kinase and Grb2, the adaptor to the guanine nucleotide exchange factor for Ras. On the other hand, no significant association was detected with two other SH2 domain proteins, the SH2-containing protein tyrosine phosphatase 2 and phospholipase Cgamma. Insulin-like growth factor I acting through its receptor was as effective as insulin in eliciting tyrosine phosphorylation of IRS-4, but interleukin 4 and epidermal growth factor were ineffective.