Childhood acute lymphoblastic leukemia: is there a tumor suppressor gene in chromosome 12p12.3?

Cytogenetic deletions on the short arm of chromosome 12 are common, recurring alterations found in a wide range of hematological neoplasias, including childhood acute lymphoblastic leukemia (ALL), the most frequent pediatric malignancy. This loss of genetic material suggests the presence of a tumor suppressor gene playing an important role in growth regulation or in the differentiation of hematopoietic stem cells. In order to substantiate this hypothesis and determine the chromosomal location of this putative gene, we and others have applied a deletion mapping strategy based on the detection of loss of heterozygosity (LOH) at specific genomic loci in leukemic cells. Hemizygous deletions at chromosome 12p12.3 were observed in childhood B cell precursor ALL, and proved to be one of the most frequent genetic alterations seen in this disease. The shortest region of overlapping deletions (SRO) was delimited by the markers D12S89 (distal) and D12S358 (proximal), separated by a genetic interval of approximately 3 cM. LOH in the same region in other hematological diseases, as well as in a variety of solid tumors, suggests either the presence of several tumor suppressor genes or the existence of a single gene with a wide range of activity. This genetic interval contains two known genes: TEL/ETV6, an ets-like transcription factor, and the cyclin-dependant kinase inhibitor, p27/kip1. Accumulating evidence suggests that an as yet unidentified tumor suppressor gene is closely linked to these two genes. Long-range restriction mapping of the SRO region allowed the construction of a approximately 750 kb physical map containing 4 known genes, 7 STSs, 3 chromosome 12 ESTs and 8 CpG islands. The construction of a 12p12.3 framework map is a crucial step towards the identification of candidate genes and should provide a valuable tool for the characterization of transcriptional units.

Allelic loss in childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most frequent cancer encountered in children. Little is known about the molecular basis of childhood ALL, although the clinical, pathological, and immunophenotypic features have been well documented. To understand the role of tumor suppressor genes (TSGs) in the development of this disease, we performed a detailed allelotype analysis. Twenty-nine patients (24 pre-B and 5 of T-cell lineage) were investigated for loss of heterozygosity (LOH), using 49 highly polymorphic markers distributed over 13 chromosomal arms which are known or postulated to contain TSGs. The highest rates of allelic losses were observed in chromosomes 9p and 12p which were deleted in 29 and 32% of the informative patients, respectively. These are among the most frequent alterations found in childhood ALL. Other losses were found at a lower frequency in chromosomes 6p, 6q, 9q, 17p, and 17q. No LOH was found at chromosomes 3p, 5q, 11p, 11q, 13q and 18q in any patient. These results suggest that many TSGs may be involved in the development of childhood ALL.

Frequent deletion of chromosome 12p12.3 in children with acute lymphoblastic leukaemia.

Cytogenetic deletions of the short arm of chromosome 12 are common recurring alterations found in a wide range of haematological neoplasias, including childhood acute lymphoblastic leukaemia (ALL), the most frequent paediatric malignancy. Such a loss of genetic material suggests the presence of a tumour suppressor gene which plays an important role in growth regulation or in the differentiation of haemopoietic stem cells. To substantiate this hypothesis and to determine more precisely the chromosomal location of this putative gene, we applied a deletion mapping strategy based on the detection of loss of heterozygosity (LOH) at specific genomic loci in tumour cells. 13 polymorphic markers were used to screen DNA samples from 20 children with ALL. LOHs at 12p12.3 were observed in almost 50% of informative B-cell precursor ALL patients analysed. This is one of the most frequent genetic alterations found in this disease. A common region of LOH was delimited by the markers D12S89 (distal) and D12S358 (proximal), separated by a genetic interval of approximately 3 cM. We refined the position of the putative 12p tumour suppressor gene to a physical interval of <1.3 Mb, a crucial step towards the identification of candidate genes. A yeast artificial chromosome clone contig that spans the entire critically deleted region includes two known genes: TEL, a member of the ets family of transcription factors, and p27KIP1, a cyclin-dependent kinase inhibitor.

Microsatellite instability in childhood T cell acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most frequent cancer encountered in children. Little is known about the molecular pathology of childhood T cell ALL. Oncogenesis is a multistep process that involves alterations in proto-oncogenes and tumor suppressor genes. Recently, a mutator phenotype detectable by microsatellite instabilities was shown to be associated with predisposition to cancer. This new mechanism for human carcinogenesis is caused by defects in the DNA replication/repair system. To study the involvement of some of these mutational events in the development of T cell ALL, we have initiated a systematic search for losses of heterozygosity (LOH) and microsatellite instabilities in children affected with this disease. These patients were allelotyped by PCR using 56 microsatellite markers located near known or putative tumor suppressor genes. The microsatellite patterns were altered in more than 80% of the patients. LOH were detected in chromosomes 6p, 12p and 9p. Two third of the patients were deleted for chromosome 9p21, suggesting the involvement of a tumor suppressor gene, probably the p16 gene. The only patient refractory to chemotherapy was shown to be associated with a mutator phenotype. This is the first documented case of a childhood neoplasia associated with genomic instabilities. Our results suggest that defects in DNA replication/repair components are involved in the development of a subset of childhood T cell ALL.

Regulation of 1,4,5-IP3, 1,3,4,5-IP4 and IP6 binding sites following entorhinal cortex lesions in rat brain.

A lesion of the entorhinal cortex produces a loss of more than 80% of the synapses in the outer molecular layer of the hippocampus in the rat. However, this synaptic loss is transient. Beginning a few days after denervation, new synapses are formed, virtually replacing the lost inputs within two months. Synaptic remodelling induced by entorhinal cortex lesion is associated with specific modifications of various neurotransmitters, hormones and growth factors. Many of these substances act at membrane bound-receptors to induce the hydrolysis of phosphatidylinositols generating various inositol phosphates. Some of the key members of this family include inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate which are all associated with the maintenance Ca2+ homeostasis. To investigate the potential roles and/or alterations of inositol phosphates in entorhinal cortex lesions-induced neuronal plasticity, we quantified specific receptor sites for inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate using their respective tritiated ligands, at different periods post-lesion corresponding to the degenerative and subsequent reinnervation phases. [3H]inositol 1,4,5-trisphosphate binding sites are maximally increased (30%) between two and eight days post-lesion in the hippocampal formation on both sides of the lesion. In the cortex, [3H]inositol 1,4,5-trisphosphate binding increased also bilaterally following the lesion. Changes in [3H]inositol 1,3,4,5-tetrakisphosphate binding are delayed and reduced (20% increase) in magnitude compared to these seen for [3H]inositol 1,4,5-trisphosphate binding. The maximal peak in [3H]inositol 1,3,4,5-tetrakisphosphate binding is observed between eight and 14 days after the lesion in the hippocampal formation and the cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Entorhinal cortex lesion induces differential responses in [125I]insulin-like growth factor I, [125I]insulin-like growth factor II and [125I]insulin receptor binding sites in the rat hippocampal formation.

The hippocampus can be induced by deafferentation to selectively reorganize its neuronal input. Entorhinal cortex lesion, which causes degeneration of the perforant pathway, evokes sprouting of septal afferents as well as glutamatergic commissural/associational fibers in the deafferentated zone of the molecular layer of the dentate gyrus. Although the process of reactive synaptogenesis that follows deafferentation has been extensively studied, at present little is known about its molecular basis and the mechanism of initiation. In this study, following unilateral lesion of the entorhinal cortex, the time-course of possible alterations of insulin-like growth factors I and II, and insulin binding sites were evaluated by in vitro quantitative receptor autoradiography. [125I]Insulin-like growth factor I receptor binding sites did not exhibit any significant variation between the contralateral and ipsilateral hippocampal formation at any time periods following lesion except in the molecular layer of the dentate gyrus (P < 0.05) at day 8. However, when compared with the unlesioned animals, a differential time-dependent response of [125I]insulin-like growth factor I binding sites was noted in selective layers of the hippocampus. [125I]Insulin-like growth factor II receptor binding sites showed a significant decrease (P < 0.05) in the ipsilateral granular cell layer of the dentate gyrus only at day 14 post lesion. Interestingly, compared to controls, a dramatic bilateral increase (P < 0.05) in [125I]insulin-like growth factor II binding was evident between days 1 and 8 in most layers of the hippocampal formation. A lesion-induced bilateral increase (P < 0.05) in [125I]insulin binding sites was evident in all layers of the hippocampus between two to eight days and at 30 days post lesion. In selective layers, however, a significant increase (P < 0.05) in [125I]insulin binding sites was also observed at days 1 and 14 after lesion. These results, which are compatible with the process of degeneration and/or sprouting of the terminal fibers, suggest possible involvement of insulin-like growth factors and insulin in the sequence of molecular events that occur to facilitate neuronal repair and to promote neuronal survival following entorhinal cortex lesion.

Cholesterol synthesis and lipoprotein reuptake during synaptic remodelling in hippocampus in adult rats.

Apolipoprotein E is synthesized and secreted by astrocytes in the hippocampus following lesions of the entorhinal cortex. It was proposed that apolipoprotein E, by analogy to its role in cholesterol transport in circulation, could be involved in the salvage and reutilization of non-esterified cholesterol released during terminal breakdown. The salvaged cholesterol could then be transported to neurons by apolipoprotein E-complexes and taken up via the apolipoprotein E/apolipoprotein B (low-density lipoprotein) receptor. To test this hypothesis, we have examined low-density lipoprotein receptor binding in brain sections of rats undergoing hippocampal reinnervation. The number of neuronal cells labelled by fluorescent Dil-low-density lipoprotein as well as the density of [125I]low-density lipoprotein binding sites in the dentate gyrus were found to increase in parallel with the extent of cholinergic reinnervation occurring in the deafferented hippocampus. In contrast, hippocampal cholesterol synthesis fell by more than 60% at eight days post-lesion, but eventually returned to control levels at 30 days post-lesion. The transient loss of cholesterol synthesis coincided with a peak in hippocampal apolipoprotein E expression. A concomitant accumulation of sudanophilic lipids (cholesterol esters and phospholipids) was detected in the outer molecular layer of the dentate gyrus and in the hilar region. The present findings suggest that non-esterified cholesterol released during terminal breakdown is esterified, transported via the apolipoprotein E transport system to neurons undergoing reinnervation, and take-up through the low-density lipoprotein receptor pathway where it is presumably used as a precursor molecule for the synthesis of new synapses and terminals.

Entorhinal cortex lesions transiently alter glucocorticoid but not mineralocorticoid receptor gene expression in the rat hippocampus.

Entorhinal cortex lesions destroy an important hippocampal input and lead to axonal sprouting in the dentate gyrus. Glucocorticoids are known to inhibit this reinnervation process. In the present study, we examined changes in hippocampal glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA expression using in situ hybridization following unilateral entorhinal cortex lesioning (ECL) in the rat. As early as 1 day postlesioning, a 33% bilateral decrease in GR mRNA expression was observed in the dentate gyrus. By contrast, a 36% bilateral increase in GR mRNA expression was detected in the CA1 cell field. GR mRNA levels in both regions returned to those of control animals 2 days postlesioning, indicating that these effects were transient. Adjacent sections hybridized with probes to MR mRNA revealed no changes in hippocampal MR gene expression as a result of ECL. The selective decrease in GR mRNA expression observed in the dentate gyrus following ECL is specific to the hippocampal subregion targeted for reactive synaptogenesis and thus may serve to attenuate the inhibitory actions of circulating glucocorticoids.

Modulation of gamma-actin and alpha 1-tubulin expression by corticosterone during neuronal plasticity in the hippocampus.

Evidence is given for altered gene expression of gamma-actin in the hippocampus in response to entorhinal cortex lesion (ECL). Time course analysis reveals a progressive repression of gamma-actin expression between 4 and 14 days post-lesion, coinciding with the early and middle phases of the hippocampal reinnervation process. RNA prevalence returns to near control values at 30 days post-lesion. Corticosterone administration, which is known to impair the reinnervation process in ECL rats, prevents the lesion-induced reduction in gamma-actin expression and blocks the induction of alpha 1-tubulin in the deafferented hippocampus. The timing of response of gamma-actin to ECL and its modulation by glucocorticoid administration support suggestions that gamma-actin has an important role to play in neuronal cytoarchitecture remodelling during hippocampal reinnervation.