Growth of tumor cells is often a function of deregulated growth factor receptors and their corresponding intracellular signalling molecules. The dissociable antibody staining arrays have the versatility to rapidly identify the expression, activation, and localization of such molecules and pathways in biopsy specimens. This report describes a protocol to quantify the activity of a panel of signalling molecules in Wilms tumor biopsy specimens and surrounding nonmalignant renal cells. We propose that this technique can be used to rapidly identify multiple markers and may aid in the study of aberrant growth regulatory mechanisms and potential targets for therapeutics from pathologic specimens.

Juvenile myelomonocytic leukemia (JMML) is considered to be one of the most difficult to treat and deadliest diseases of early childhood. The pathogenesis of JMML involves deregulated cytokine signal transduction, especially a selective hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Current treatment modalities do not provide significant long-term disease control. We describe an experimental model system in which the effect of targeted agents can be studied in the context of GM-CSF mediated growth and survival of JMML cells. Bone marrow and peripheral mononuclear cells from JMML patients were enriched by immunoaffinity chromatography and grown in the presence of GM-CSF. Purity of these cells was confirmed by FACS and their absolute dependence on GM-CSF for growth and survival was assessed. A direct role for GM-CSF was confirmed by the addition of neutralizing antibodies. Molecular techniques showed the lack of excess GM-CSF secretion by these cells that proved receptor hypersensitivity as the principal driving force behind the excessive proliferation. To understand the signaling pathways involved in this process, we constructed a capture antibody array with antibodies to critical signaling molecules. GM-CSF treated and control lysates were incubated with the arrays and the expression and phosphorylation of the signaling molecules were detected by anti p-tyr, p-Ser and p-Thr antibodies. Blots were then quantified by an automated image analysis program. Results showed consistent activation of a group of signaling molecules mediated by the addition of GM-CSF. These include IRS-1, FAK, IkappaB and LIFR. Using this assay system we then looked at the effects of a number of novel therapeutic agents to inhibit the growth and specifically interfere with the GM-CSF mediated signaling cascade. These novel therapeutic agents and targeted kinase inhibitors such as 17-AAG, DMAG, arsenic trioxide, rebeccamycin, AMN-107, and Prima-1. (IC50: 0.1, 0.01. 0.5, 0.1, 0 and 10, uM respectively). These agents were studied in combination to identify agents that exhibit synergistic and additive properties. Significant additive effects were seen with certain combinations, notably in combination with Hsp90 inhibitors. We describe an effective experimental model to identify the players and pathways involved in the abnormal growth properties of JMML. We discuss in detail the implications of these findings in the development of potential anti JMML therapies in the future.

Granulosa cell tumors (GCTs) of the ovary are relatively rare and account for <5% of all ovarian cancers. The molecular pathogenesis of these tumors is not well understood. We tested the hypothesis that cyclin‐dependent kinase inhibitors, specifically the in hibitors of the cyclin‐dependent k inase 4 (INK4) family, are targets for altered gene expression in GCTs. The status of RB1, INK4A, INK4B, INK4C, INK4D, and ARF in 13 adult and 2 juvenile ovarian GCTs was determined by reverse transcription–polymerase chain reaction of total RNA and exon‐specific sequencing of genomic DNA. Tumors showing loss of INK4A expression were assayed further by exon‐deletion analysis and methylation‐specific PCR. None of the juvenile tumors demonstrated altered expression, but 7/12 (58%) adult GCTs lacked expression of INK4A, INK4B, or both. In one of these cases, we noted a homozygous deletion of the INK4A locus, and in the remaining tumors we found hypermethylation of the promoter region, a mechanism that can lead to gene inactivation. These data support a role for the INK4 family of CDK inhibitors in the biology of GCTs. © 2002 Wiley‐Liss, Inc.

Cyclin-dependent kinase (CDK) inhibitors represented by the INK4 family (including p16(INK4a), (CDKN2A), p15(INK4b), (CDKN2B) p18(INK4c,CDKN2C), and p19(INK4d), (CDKN2D)) are regulators of the cell cycle shown to be aberrant in many types of human cancer. We tested the hypothesis that these CDK inhibitors are a target for altered gene expression in Wilms tumor. Using RT-PCR, gene expression of the INK4 family was found to be decreased in 9 of 38 Wilms tumor samples obtained from the National Wilms Tumor Study Group (NWTSG) tissue bank. All the affected tumor samples were of favorable histology, Methylation-specific PCR revealed that methylation in the p16 promoter region may be responsible for altered expression. The incidence of loss of p16 expression may increase with increasing tumor stage, i.e., 1/10 (10%) with stage I/II FH Wilms tumor, 2/10 (20%) with stage III FH Wilms tumor, and 4/10 (40%) with stage IV FH Wilms tumor. Thus, determining the expression status of the INK4 Family may have potential prognostic value in the management of Wilms tumor. Genes Chromosomes Cancer 29:63-69, 2000. (C) 2000 Wiley-Liss, Inc.

BACKGROUNDPlatinum agents are frequently combined with ifosfamide. Mesna, originally coadministered to protect from ifosfamide side effects, might also react with the platinum agents in these combinations.METHODSMalignant glioma cells were incubated with cisplatin, carboplatin and mesna. Cell numbers were measured by counting and by MTT-tests.RESULTSIn cell free solution mesna turned MTT to its blue farmazan product. Mesna's effect on cells were cell-line specific: It penetrated U87 cells without effect on growth, reduced cell numbers in C6 and T98G cells and did not alter U251 cells. The concentration of cisplatin killing 50% of the cells were 7 x 10(-7) in C6, 9.7 x 10(-6) in T98G, 1.2 x 10(-5) in U251 and 2.4 x 10(-4) in U87 cells. For the same effect, carboplatin required 3-10 times higher concentrations. Mesna protected all cell lines from the cytotoxicity of the platinum agents.CONCLUSIONClinical studies should specify in detail, infusion schedules of mesna and platinum agents.