Phase II study of arsenic trioxide and ascorbic acid for relapsed or refractory lymphoid malignancies: a Wisconsin Oncology Network study.

Arsenic trioxide (As(2)O(3)) has established clinical activity in acute promyelocytic leukaemia and has pre-clinical data suggesting activity in lymphoid malignancies. Cell death from As(2)O(3) may be the result of oxidative stress. Agents which deplete intracellular glutathione, such as ascorbic acid (AA), may potentiate arsenic-mediated apoptosis. This multi-institution phase II study investigated a novel dosing schedule of As(2)O(3) and AA in patients with relapsed or refractory lymphoid malignancies. Patients received As(2)O(3) 0.25 mg/kg iv and AA 1000 mg iv for five consecutive days during the first week of each cycle followed by twice weekly infusions during weeks 2-6. Cycles were repeated every 8 weeks. The primary end point was objective response. In a subset of patients, sequential levels of intracellular glutathione and measures of Bcl-2 and Bax gene expression were evaluated in peripheral blood mononuclear cells during treatment. Seventeen patients were enrolled between March 2002 and February 2004. The median age was 71, and the majority of enrolled patients had non-Hodgkin's lymphoma (12/17). Sixteen patients were evaluable, and one patient with mantle cell lymphoma achieved an unconfirmed complete response after five cycles of therapy for an overall response rate of 6%. The trial, which had been designed as a two-stage study, was closed after the first stage analysis due to lack of activity. Haematologic toxicities were the most commonly reported events in this heavily pre-treated population, and comprised the majority of grade 3 and 4 toxicities. Intracellular depletion of glutathione was not consistently observed during treatment. As(2)O(3) and AA in this novel dosing strategy was generally well tolerated but had limited activity in patients with relapsed and refractory lymphoid malignancies.

The spectrum of molecular alterations in the evolution of chronic myelocytic leukemia.

DNA from 135 patients with chronic myelogenous leukemia (CML) at various clinical stages and Philadelphia (Ph1) chromosome positive acute lymphoblastic leukemia was investigated for alterations in a variety of proto-oncogenes which have been implicated in the evolution of CML from its chronic phase to blast crisis. The most common genetic change found in the evolution of typical Ph1 chromosome positive CML to blast crisis was an alteration of the p53 gene involving either a rearrangement, a deletion, or a point mutation in the coding sequence of the gene. Alterations of the p53 gene were found in the myeloid and the rare megakaryocytic variant of blast crisis but were absent in the lymphoid leukemic transformants. Gross structural alterations were seen in 11 of 54 (20%) of myeloid or unknown phenotypes of blast crisis and in only 1 of 44 chronic phase cases. Eight examples of mutations in the open reading frame of the p53 gene at codons 49, 53, 60, 140, 202, 204, 238, and 239 were observed in blast crisis patients. Mutations in the N-RAS gene were rare in typical blast crisis (2 of 27 cases) but were found in megakaryocytic and Ph1 negative myeloid blast crisis. We concluded that heterogeneous alterations in the p53 gene and occasionally in the N-RAS genes accompany the evolution of chronic phase CML to blast crisis.

t(9;11)(p22;p15) in acute myeloid leukemia results in a fusion between NUP98 and the gene encoding transcriptional coactivators p52 and p75-lens epithelium-derived growth factor (LEDGF).

A t(9;11)(p22;p15) chromosomal translocation was identified in an adult patient with de novo acute myelogenous leukemia. Fluorescence in situ hybridization and Southern blot analysis mapped the 11p15 break-point to the NUP98 gene. Using 3' rapid amplification of cDNA ends, we have identified a chimeric mRNA that fused the NUP98 FXFG repeats in frame to the COOH-terminal portion of the gene encoding the transcriptional coactivators p52 and p75, also known as lens epithelium-derived growth factor (LEDGF). As expected, both NUP98-p52 and NUP98-p75 (LEDGF) chimeric mRNAs were detected by reverse transcription-PCR; however, the reciprocal p52/p75 (LEDGF)-NUP98 fusion mRNA was not detected. Our results demonstrate that this is the most 5' NUP98 fusion reported and reveal a previously unrecognized genetic target, the gene encoding p52/p75 (LEDGF).

Proto-oncogene abnormalities in human breast cancer: c-ERBB-2 amplification does not correlate with recurrence of disease.

The incidence of c-ERBB-2 amplification in breast cancers and its usefulness as a predictor of tumor recurrence after treatment have been subjects of controversy (Ali et al., 1988, Slamon et al., 1987). We re-examined this subject by analysing 157 primary and 14 metastatic breast cancers with c-ERBB-2 and 18 other molecular probes as controls. Five proto-oncogenes were found to be occasionally amplified in primary breast cancers: c-ERBB-2 (11%), c-MYB (3%), c-RAS-Ki (3%), INT-2 (4%) and c-MYC (6%). No statistically significant correlation between amplification of c-ERBB-2 and recurrence of tumors was observed. The only significant correlation observed was between early recurrence of advanced (stage III) tumors and amplification of one or another of the above five proto-oncogenes. We conclude that breast cancer utilize multiple genetic mechanisms in their progression and metastasis, and that analysis of c-ERBB-2 alone is not a useful guide.

Variation in the protein coding region of the human p53 gene.

This report describes a restriction fragment length polymorphism for the enzyme BglII caused by a conserved C to T change at residue 21 of the human p53 gene. This RFLP could potentially be misinterpreted as a rearrangement or a point mutation if paired constitutional tissue is not simultaneously analyzed.

Expression of the candidate Wilm's tumor gene, WT1, in human leukemia cells.

Wilms' tumor (WT) is a pediatric malignancy that occurs in embryonic kidney. Recently, a putative Wilms' tumor gene (WT1), located on chromosome 11p13, was isolated and characterized. We found constitutive expression of WT1 mRNA in eight out of 22 hematopoietic cell lines and seven out of 26 clinical samples which were derived from patients with various types of hematologic malignancies. WT1 mRNA was detected in four out of six myeloid cell lines, four out of 10 cases of acute myelocytic leukemia, three out of 15 lymphoid cell lines, one out of nine cases of lymphoid malignancies, and one out of six cases of chronic myelocytic leukemia in accelerated phase and blast crisis. One unclassified hematopoietic cell line and a case of myelodysplastic syndrome also expressed WT1 mRNA. No mutations were detectable in the cell lines by Southern blot analysis and a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis in the four zinc finger domains of the WT1 gene. These results suggest that WT1 gene is expressed in several types of immature lymphoid or myeloid leukemia cells possibly without alterations of the WT1 gene.

Differential expression of clusterin in the testis and epididymis of postnatal and germ cell deficient mice.

Clusterin is found in a wide variety of tissues and is expressed in a number of physiological and pathological contexts. It is expressed constitutively in the adult male reproductive tract, specifically the testes and caput of the epididymis. The gene is also induced during apoptotic cell death in the kidney and hormone-dependent tissues such as the prostate and mammary gland. The mechanisms controlling the expression of clusterin gene expression in these tissues are still unknown, although it has been suggested that interactions between the Sertoli cells and germ cells in the testis, or epithelial cells and germ cells in the epididymis, may be required for clusterin expression. To investigate the importance of germ cells in the induction of clusterin expression in these tissues, we have used in situ hybridization to determine the developmental regulation and germ cell dependence of clusterin expression in the reproductive tract of the normal and germ cell deficient male mice. Clusterin mRNA is present in the supporting cells in the testes of normal mice and in both atrichosis (at) and dominant spotting mutant (Wv) mice, both of which are germ cell deficient. On the other hand, the expression of clusterin in the epididymis appears to be at least partially dependent of the presence of germ cells, because segmental localization of clusterin mRNA within the caput of the epididymis that is seen in normal mice is disrupted in the at and Wv mutants. In these germ cell deficient mutants, the steady-state level of clusterin mRNA, which is repressed in segment 1 and 2 of the adult wild type mouse, is expressed at levels similar to those seen at days 7 and 17 during the development of the normal epididymis.

The retinoid X receptor and its ligands: versatile regulators of metabolic function, cell differentiation and cell death.

Retinoid X Receptors (RXRs) consist of a family of nuclear receptors that target and regulate multiple signalling pathways. The early evolutionary emergence of RXRs in comparison to other nuclear receptors may have allowed for the development of unique properties as transcriptional regulators. Indeed, the complexity of these receptors is derived from their ability to activate transcription as homodimers or as obligate heterodimeric partners of a multitude of other nuclear receptors. In addition, RXRs can regulate gene expression in a ligand-dependent (forming permissive heterodimeric complexes) or - independent (forming non-permissive heterodimeric complexes) manner. Given that ligand binding is a critical component of RXR function, this review will focus on the ligand dependent functions of RXR. The remarkably conserved ligand binding domain of RXR is a multi-functional structure that in addition to ligand binding, serves as a homo- and heterodimeric interface, and a region to bind coactivactor and corepressor molecules. RXRs have a small ligand binding pocket and therefore bind their ligands (such as 9-cis RA) with both high affinity and specificity. In the presence of ligand, permissive RXR heterodimers bind coactivators, but nonpermissive complexes can bind coactivators or corepressors depending on the activation of the RXR's heterodimeric partner. Physiologically, the temporal and tissue specific pattern of RXRs as well as the presence of phenotypic abnormalities in receptor knockout studies (most severe in RXRa -/- animals) demonstrate the important role for these receptors both during development (morphogenesis) and in adult differentiated tissues (cell proliferation, cell differentiation, cell death). These receptors also play an important regulatory role metabolic signaling pathways (glucose, fatty acid and cholesterol metabolism), including metabolic disorders such as type 2 diabetes, hyperlipidemia and atherosclerosis. RXRs function as master regulators producing diverse physiological effects through the activation of multiple nuclear receptor complexes. RXRs represent important targets for pharmacologic interventions and therapeutic applications.

Cell death/apoptosis: normal, chemically induced, and teratogenic effect.

Cell death is an integral part of a variety of biological processes including cell proliferation, differentiation, and morphogenesis. We review here the morphological and biochemical nature as well as the genetic basis for cell death during normal and abnormal development. Most often referred to in normal development as programmed cell death, this controlled process determines the size, patterning, and function of many tissues. The importance of its proper genetic regulation is demonstrated by the discovery of cell death-specific genes and the several disorders including cancer and teratogenesis that result from repression or enhancement of cell death. In our studies we employed the developing mouse limb, which provides a defined window of active cell death, to elucidate mechanisms of cell death. We have developed markers that reveal in the developing normal limb an apoptotic morphology with phagocytosis and DNA fragmentation. In the limb deformity mutant Hammertoe there is a defective (restricted) cell death pattern, but the morphology remains apoptotic. By the use of these markers, we were able to observe that the teratogen retinoic acid produced enhanced apoptotic cell death. Most interestingly, retinoic acid-induced cell death in the Hammertoe mutant resulted in correction of the mutant phenotype. Future studies will determine the relationship between exogenous agents and endogenous signaling pathways as well as indicate how these interactions can alter the fate of a given cell and potentially ameliorate a genetic abnormality.

Genetic and cytogenetic changes in acute lymphoblastic leukemia.

In recent years, a number of non-random chromosomal alterations have been identified in specific populations of acute lymphoblastic leukemic cells of either B-cell or T-cell lineage. The most frequently involved chromosomal sites are 1q, 4q, 6q, 7q, 8q, 9p, 9q, 10q, 11p, 12p, 14q, 19p and 22q. Genes located near frequent breakpoints include c-myc, c-abl and the genes for the T-cell alpha and beta receptors. In addition, approximately 20 other genes potentially involved in the leukemic process are located near less frequently encountered, but consistent, chromosomal breakpoints.

Apoptotic cell death in the limb and its relationship to pattern formation.

Detection of cell death throughout embryogenesis demonstrates its importance in the normal form and function of the organism. We have examined cellular death during normal limb development by use of markers that display the morphology of cell death, the presence of phagocytic cells, and lysosomal activity. In addition in situ labeling confirms fragmentation of DNA in the mammalian limb. By these criteria, cell death in the developing limb can be categorized as type 1 or apoptotic cell death. However, the signal(s) responsible for cellular destruction and activation of phagocytosis by neighboring cells or recruited macrophages remain to be identified. The decision for cellular fate during development and regulation of it once the decision is made are key questions. To address the specific question of what determines that one cell will die while its neighbor survives, we have used compounds, such as retinoic acid (RA), that have been shown to alter the pattern of normal development. We and others have shown that RA does indeed alter the pattern of cell death to the extent of inducing malformations in the limb. The mouse mutant Hammertoe (Hm) provides an abnormal system in which the pattern of cell death is specifically altered in the interdigital regions of the limb. Our preliminary data suggest that RA can also introduce cell death between digits 2, 3, 4, and 5 of the Hm mutant where there was no cell death to begin with. Our observations of the effect of RA on mutant limbs suggest that a direct relationship between RA and cell death does exist and that this interaction may be required for correct pattern formation. The alteration in the pattern of cell death in the mutant mouse is of great interest, since it would provide a rare example of specific correction of a birth defect by direct intercession against the physiological effect of the mutation.