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Biomedical subjects

M A Israel

Publications and source records attributed to M A Israel.

At least 37 records · Page 2Linked to original sources
PubMed1999

Id-1 and Id-2 are overexpressed in pancreatic cancer and in dysplastic lesions in chronic pancreatitis.

Id proteins antagonize basic helix-loop-helix proteins, inhibit differentiation, and enhance cell proliferation. In this study we compared the expression of Id-1, Id-2, and Id-3 in the normal pancreas, in pancreatic cancer, and in chronic pancreatitis (CP). Northern blot analysis demonstrated that all three Id mRNA species were expressed at high levels in pancreatic cancer samples by comparison with normal or CP samples. Pancreatic cancer cell lines frequently coexpressed all three Ids, exhibiting a good correlation between Id mRNA and protein levels, as determined by immunoblotting with highly specific anti-Id antibodies. Immunohistochemistry using these antibodies demonstrated the presence of faint Id-1 and Id-2 immunostaining in pancreatic ductal cells in the normal pancreas, whereas Id-3 immunoreactivity ranged from weak to strong. In the cancer tissues, many of the cancer cells exhibited abundant Id-1, Id-2, and Id-3 immunoreactivity. Scoring on the basis of percentage of positive cells and intensity of immunostaining indicated that Id-1 and Id-2 were increased significantly in the cancer cells by comparison with the respective controls. Mild to moderate Id immunoreactivity was also seen in the ductal cells in the CP-like areas adjacent to these cells and in the ductal cells of small and interlobular ducts in CP. In contrast, in dysplastic and atypical papillary ducts in CP, Id-1 and Id-2 immunoreactivity was as significantly elevated as in the cancer cells. These findings suggest that increased Id expression may be associated with enhanced proliferative potential of pancreatic cancer cells and of proliferating or dysplastic ductal cells in CP.

Adolescent↗
PubMed1998

Injury selectively down-regulates the gene encoding for the Id4 transcription factor in primary cultures of forebrain astrocytes.

Astrogliosis is an important component of the response to injury of the central nervous system (CNS). The Id family of helix-loop-helix (HLH) transcription factors has been implicated in the regulation of cellular differentiation in several different lineages and may contribute to the regulation of astrogliosis. We examined the expression of Id genes in primary cultures of mouse forebrain astrocytes under experimental conditions in which astrogliosis was elicited by mechanical injury. Astrocyte cultures expressed the four known members of the Id gene family, Id1, Id2, Id3, and Id4. After injury, at a time when astrocytes developed the characteristic phenotypic changes of astrogliosis, Id4 expression decreased dramatically. Id1, Id2, and Id3 mRNA levels did not change. These results identify Id4 as a candidate marker of astroglial activation in culture and suggest that Id4 expression plays a role in the process of astrogliosis.

Animals↗
PubMed1998

The intrinsic radioresistance of glioblastoma-derived cell lines is associated with a failure of p53 to induce p21(BAX) expression.

Radiation is the primary modality of therapy for all commonly occurring malignant brain tumors, including medulloblastoma and glioblastoma. These two brain tumors, however, have a distinctly different response to radiation therapy. Medulloblastoma is very sensitive to radiation therapy, whereas glioblastoma is highly resistant, and the long-term survival of medulloblastoma patients exceeds 50%, while there are few long-term survivors among glioblastoma patients. p53-mediated apoptosis is thought to be an important mechanism mediating the cytotoxic response of tumors to radiotherapy. In this study, we compared the response to radiation of five cell lines that have wild-type p53: three derived from glioblastoma and two derived from medulloblastoma. We found that the medulloblastoma-derived cell lines underwent extensive radiation-induced apoptotic cell death, while those from glioblastomas did not exhibit significant radiation-induced apoptosis. p53-mediated induction of p21(BAX) is thought to be a key component of the pathway mediating apoptosis after the exposure of cells to cytotoxins, and the expression of mRNA encoding p21(BAX) was correlated with these cell lines undergoing radiation-induced apoptosis. The failure of p53 to induce p21(BAX) expression in glioblastoma-derived cell lines is likely to be of biologic significance, since inhibition of p21(BAX) induction in medulloblastoma resulted in a loss of radiation-induced apoptosis, while forced expression of p21(BAX) in glioblastoma was sufficient to induce apoptosis. The failure of p53 to induce p21(BAX) in glioblastoma-derived cell lines suggests a distinct mechanism of radioresistance and may represent a critical factor in determining therapeutic responsiveness to radiation in glioblastomas.

Apoptosis↗
PubMed1998

The mouse Id2 and Id4 genes: structural organization and chromosomal localization.

The Id proteins belong to a family of nuclear HLH proteins lacking a basic region and thought to function as dominant-negative regulators of bHLH proteins during cell growth and differentiation. In this paper, we report the genomic organization of the mouse Id2 and Id4 genes. These genes each span approximately 3 kb of the mouse genome and are each organized as three exons with recognizable splice donor and acceptor consensus sequences. Their genomic organization is very similar, consistent with their having evolved from a common, ancestral Id-like gene. Using FISH analysis, we have localized the mouse Id2 and Id4 genes to mouse chromosome 12 and 13, respectively.

Amino Acid Sequence↗
PubMed1998

The helix-loop-helix protein Id2 is overexpressed in human pancreatic cancer.

Id2 belongs to the Id family of helix-loop-helix (HLH) proteins, which upon heterodimerization with basic HLH proteins prevent basic HLH proteins from DNA binding. Proteins of the Id family act as negative regulatory transcriptional factors, and their expression correlates with cell proliferation and arrested differentiation in many cell lineages. In this study, we characterized the expression of Id2 in normal and cancerous pancreatic tissues. Pancreatic cancers markedly overexpressed Id2 mRNA in comparison to the normal pancreas. Furthermore, there was abundant Id2 immunoreactivity in the cancer cells within the pancreatic tumor mass. In PANC-1 human pancreatic cancer cells, steady-state Id2 mRNA levels increased upon serum addition and decreased after induction of differentiation with either sodium butyrate or 12-O-tetradecanoylphorbol-13-acetate. Inhibition of Id2 expression with Id2 antisense oligonucleotides inhibited the growth of these cells, whereas random and sense oligonucleotides were without effect. These findings suggest that Id2 may have a role in human pancreatic cancer.

Adolescent↗
PubMed1998

Ionizing radiation inhibits chemotherapy-induced apoptosis in cultured glioma cells: implications for combined modality therapy.

Surgical resection followed by radiation therapy is the mainstay of treatment for glioblastoma multiforme (GBM), the most aggressive of the malignant gliomas. The poor clinical response of GBM and the intrinsic radiation resistance of this tumor type have prompted clinical investigations seeking to define the role of chemotherapy in the treatment of GBM. In this study, we examined the cytotoxic response of GBM-derived cell lines to treatment with both radiation and chemotherapy. We observed that the sensitivity of glioma cells to cisplatin- and FAS-induced apoptosis was diminished by prior treatment with ionizing radiation. Radiation conferred resistance to cisplatin and FAS cytotoxicity in a dose- and time-dependent manner. Radiation diminished the cisplatin-induced cytotoxicity of malignant glioma cells but failed to alter the cisplatin susceptibility of normal primary human astrocytes. Given the role of p53 in the response of cells to irradiation, we evaluated whether p53 function affects the observed radiation-induced resistance to cisplatin. By examining isogenic cell lines differing only in p53 function, we demonstrated that radiation conferred resistance to cisplatin independently of p53. Current clinical strategies in the treatment of astrocytic tumors, which include combined modality therapy, have been empirically derived from limited clinical experience. Further understanding of the molecular determinants of apoptosis associated with combined modality therapy may guide the design of more efficacious multimodality protocols.

Apoptosis↗
PubMed1998

Cloning of human ENC-1 and evaluation of its expression and regulation in nervous system tumors.

We recently identified and characterized a novel murine gene, ENC-1, that is expressed primarily in the nervous system and encodes an actin-binding protein. To gain insight into a potential role for ENC-1 gene in the processes of cell differentiation and malignant transformation in the human nervous system, we first cloned and characterized the human homologue of ENC-1. The human ENC-1 gene appeared to be highly expressed in adult brain and spinal cord, and in a number of cell lines derived from nervous system tumors we detected low steady-state levels of ENC-1 mRNA. We used a neuroblastoma differentiation model, the retinoic acid-induced neuronal differentiation of SMS-KCNR cells, to study the regulation of the ENC-1 gene during neural crest cell differentiation. We found that the expression of ENC-1 increased dramatically in the differentiated SMS-KCNR cells as compared to control undifferentiated cells. These results suggest that ENC-1 expression plays a role during differentiation of neural crest cells and may be down regulated in neuroblastoma tumors.

Amino Acid Sequence↗
PubMed1998

Id2 promotes apoptosis by a novel mechanism independent of dimerization to basic helix-loop-helix factors.

Members of the helix-loop-helix (HLH) family of Id proteins have demonstrated roles in the regulation of differentiation and cell proliferation. Id proteins inhibit differentiation by HLH-mediated heterodimerization with basic HLH transcription factors. This blocks their sequence-specific binding to DNA and activation of target genes that are often expressed in a tissue-specific manner. Id proteins can also act as positive regulators of cell proliferation. The different mechanisms proposed for Id-mediated promotion of entry into S phase also involve HLH-mediated interactions affecting regulators of the G1/S transition. We have found that Id2 augments apoptosis in both interleukin-3 (IL-3)-dependent 32D.3 myeloid progenitors and U2OS osteosarcoma cells. We could not detect a similar activity for Id3. In contrast to the effects of Id2 on differentiation and cell proliferation, Id2-mediated apoptosis is independent of HLH-mediated dimerization. The ability of Id2 to promote cell death resides in its N-terminal region and is associated with the enhanced expression of a known component of the programmed cell death pathway, the proapoptotic gene BAX.

Animals↗
PubMed1997

ENC-1: a novel mammalian kelch-related gene specifically expressed in the nervous system encodes an actin-binding protein.

We have identified and characterized a novel murine gene, Ectoderm-Neural Cortex-1 (ENC-1), that is an early and highly specific marker of neural induction in vertebrates. ENC-1, which encodes a kelch family related protein, is expressed during early gastrulation in the prospective neuroectodermal region of the epiblast and later in development throughout the nervous system (NS). ENC-1 expression is highly dynamic and, after neurulation, preferentially defines prospective cortical areas. The only apparent expression of ENC-1 outside the NS is restricted to the rostral-most somitomere of the presomitic mesoderm, at the times corresponding to the epithelialization that precedes somite formation. Cellular expression of epitope-tagged ENC-1 shows extensive co-localization of ENC-1 with the actin cytoskeleton, and immunoprecipitation studies demonstrate a physical association between ENC-1 and actin. ENC-1 functions as an actin-binding protein that may be important in the organization of the actin cytoskeleton during neural fate specification and development of the NS.

Amino Acid Sequence↗
PubMed1997

Id genes encoding inhibitors of transcription are expressed during in vitro astrocyte differentiation and in cell lines derived from astrocytic tumors.

Id proteins belong to a class of nuclear transcription factors known as helix-loop-helix proteins. It has been reported that Id genes function as negative regulators of differentiation, and Id gene expression is down-regulated during cell differentiation. We examined the regulation of Id genes during astrocyte differentiation in a murine nervous system precursor cell line, NSEHip2-28, which is able to differentiate along the astroglial lineage, as well as in human astroglial tumor cell lines. Upon induction of NSEHip2-28 differentiation, at a time when glial fibrillary acidic protein expression became detectable, the expression of all four Id family members initially increased dramatically, and subsequently decreased. Furthermore, varying levels of Id gene expression were found in astroglial tumor cell lines displaying variable degrees of lineage-specific differentiation. These results suggest that the expression of Id family members may play an important role in the control of astrocyte differentiation.

Animals↗
PubMed1997

P16 deletion and mutation analysis in human brain tumors.

We screened human primary and recurrent malignant glioma, juvenile pilocytic astrocytoma, medulloblastoma, and meningioma tissue specimens for alterations in p16 gene structure. Single strand conformation polymorphism (SSCP) analysis was used to screen for point mutations, and a quantitative polymerase chain reaction-based assay was used to screen for homozygous gene deletions. In malignant glioma specimens, homozygous p16 gene deletions were significantly more common in high-grade tumors than in low-grade gliomas. Point mutations causing alteration in predicted protein structure were not detected. Medulloblastomas showed rare homozygous deletions and no point mutations. No mutations were detected in meningiomas.

Carrier Proteins↗
PubMed1996

RTVP-1, a novel human gene with sequence similarity to genes of diverse species, is expressed in tumor cell lines of glial but not neuronal origin.

A novel gene, RTVP-1, which shows significant sequence identity to the mammalian testis-specific proteins, a family of plant pathogenesis-related proteins and the vespid venom allergen, antigen-5, has been isolated from a cDNA library of the human glioblastoma brain tumor cell line, U-251 MG. The highest degree of sequence identity was with the human testis-specific protein, TPX1 (38.7% over 119 amino acids). Northern hybridization analysis revealed that in fetal tissue RTVP-1 RNA was detected only in the kidney, but its expression was ubiquitous in adult tissues including brain. Multiple mRNAs encoded by RTVP-1 were highly expressed in a panel of cell lines from nervous system tumors arising from glia, although expression was low or absent in nonglial-derived nervous system tumor cell lines.

Amino Acid Sequence↗
PubMed1996

Adaptation of a simple flow cytometric assay to identify different stages during apoptosis.

Apoptosis occurs under many different physiological and pathological conditions, and it reflects a genetically encoded suicide program that can be triggered by different stimuli in susceptible cells. We adapted a flow cytometric assay for the detection of apoptosis based on differential staining of viable cells with two different DNA binding dyes, propidium iodide (PI) and Hoechst 33342 (Ho342). Apoptosis was induced in different cell lines by gamma irradiation, an anti-FAS monoclonal antibody, or ganciclovir in Herpes simplex virus-thymidine kinase-expressing cells. We could identify three different populations that appeared sequentially after the induction of apoptosis. Cells corresponding to these populations were sorted and assessed for evidence of apoptosis as determined by alterations of nuclear morphology and detection of endonucleolytic activity. This analysis revealed a PI- population with subtle apoptotic changes and increased Ho342 fluorescence compared with untreated cells. Extensive apoptotic alterations were observed in a PI+ population that increased over time following the induction of apoptosis. A third population was characterized by an intermediate intensity of PI fluorescence and decreased Ho342 fluorescence compared with the other populations. This population appeared late after treatment and consisted of apoptotic bodies. Taken together, these data suggest that distinct stages of apoptosis can be identified by differential staining of cells with Ho342 and PI. This assay should be useful for the detection and further characterization of cells at different stages in the apoptotic process.

Apoptosis↗
PubMed1996

p53-dependent G1 arrest and p53-independent apoptosis influence the radiobiologic response of glioblastoma.

PURPOSE: Loss of the p53 tumor suppressor gene has been associated with tumor progression, disease relapse, poor response to antineoplastic therapy, and poor prognosis in many malignancies. We have investigated the contribution of p53-mediated radiation-induced apoptosis and G1 arrest to the well described radiation resistance of glioblastoma multiforme (GM) cells. METHODS AND MATERIALS: Radiation survival in vitro was quantitated using linear quadratic and repair-saturation mathematical models. Isogenic derivatives of glioblastoma cells differing only in their p53 status were generated using a retroviral vector expressing a dominant negative mutant of p53. Radiation-induced apoptosis was assayed by Fluorescence-activated cell sorter (FACS) analysis, terminal deoxynucleotide transferase labeling technique, and chromatin morphology. Cells were synchronized in early G1 phase and mitotic and labeling indices were measured. RESULTS: Radiation-induced apoptosis of GM cells was independent of functional wild-type p53 (wt p53). Decreased susceptibility to radiation-induced apoptosis was associated with lower alpha values characterizing the shoulder of the clonogenic radiation survival curve. Using isogenic GM cells differing only in their p53 activity, we found that a p53-mediated function, radiation-induced G1 arrest, could also influence the value of alpha and clonogenic radiation resistance. Inactivation of wt p53 function by a dominant negative mutant of p53 resulted in a significantly diminished alpha value with no alteration in cellular susceptibility to radiation-induced apoptosis. The clonal derivative U87-LUX.8 expressing a functional wt p53 had an alpha (Gy-1) value of 0.609, whereas the isogenic clonal derivative U87-175.4 lacking wt p53 function had an alpha (Gy-1) value of 0.175. CONCLUSION: We conclude that two distinct cellular responses to radiation, p53-independent apoptosis and p53-dependent G1-arrest, influence radiobiological parameters that characterize the radiation response of glioblastoma cells. Further understanding of the molecular basis of GM radiation resistance will lead to improvement in existing therapeutic modalities and to the development of novel treatment approaches.

Apoptosis↗
PubMed1996

Molecular cloning of the cDNA encoding a helix-loop-helix protein, mouse ID1B: tissue-specific expression of ID1A and ID1B genes.

A cDNA encoding a 168 amino acid mouse ID1B helix-loop-helix protein, the longest among the ID family of proteins so far identified, was cloned and its nucleotide sequence determined. Mouse ID1B mRNA is distinguishable from the mRNA encoding ID1A at its 3' end, and the relative level of expression of these two different mRNAs is similar in most tissues, with exception of skeletal muscle and kidney. Comparison of the most carboxyl terminal predicted amino acid sequences of ID1 proteins reveals 100% identity for ID1A, but the predicted Id1B proteins of several species are different.

Alternative Splicing↗
PubMed1996

Manganese superoxide dismutase expression in human central nervous system tumors.

Manganese superoxide dismutase (MnSOD) is a superoxide anion scavenger located in mitochondria. Increased expression of MnSOD can diminish oxygen radical-mediated injuries and the cytotoxic effects of tumor necrosis factor alpha, ionizing radiation, and certain chemotherapeutic agents. We used immunohistochemical staining to analyze 42 specimens of human brain tumors and 3 normal brain controls with a polyclonal antibody recognizing human MnSOD. We measured MnSOD in cerebrospinal fluid (CSF) from 14 patients with brain tumors and 7 control patients using an ELISA. Although MnSOD is not readily detected in normal brain, malignant central nervous system tumors, including tumors metastatic to the brain, displayed marked immunoreactivity to MnSOD intracellularly, in the extracellular matrix and in the tumor endothelial cells. Grade IV astrocytomas (glioblastomas), Grade III astrocytomas, and medulloblastomas were strongly immunoreactive, whereas Grade II astrocytomas had much less immunoreactivity. ELISA analysis of CSF samples from patients with malignant tumors also revealed high levels of MnSOD protein, up to 45-fold greater than the level of control CSF samples.

Brain↗
PubMed1996

Id2 expression increases with differentiation of human myeloid cells.

Id proteins are helix-loop-helix (HLH) transcriptional factors that lack the basic DNA binding domain. The Id proteins have been reported generally to function as inhibitors of cell differentiation, and their gene expression is often downregulated during cell differentiation. We examined the expression of human Id mRNAs by Northern hybridization in 11 human myeloid cell lines, several myeloid cell lines induced to differentiate, fresh myeloid leukemia samples, and normal human myeloid cells. Id2 mRNA was expressed in myelomonoblastic and monoblastic leukemic cells (PLB-985, THP-1, and U-937) but was weakly expressed in myeloblastic leukemic cells (KG-1 and HL-60). Id2 mRNA levels markedly increased with induction of differentiation of myeloid blasts (HL-60, PLB-985, THP-1, and U-937) toward either granulocytes or macrophages. Examination of fresh acute myeloid leukemic samples from 22 individuals also showed prominent Id2 mRNA expression in those samples having more differentiated blasts. Using the French-American-British classification, only 2 of 8 M0/M1 samples expressed Id2 mRNA; however, 10 of 13 M2/M3/M4 samples expressed it. In normal human myeloid cells, Id2 mRNA was expressed in cultured macrophages from bone marrow and in mature granulocytes and monocytes from peripheral blood. The half-life of Id2 mRNA was short (1 hour), and its expression was inducible by cessation of protein synthesis. Id3 mRNA was moderately expressed in monoblastic cell lines (THP-1 and U-937), and levels decreased with their differentiation. Almost no Id3 expression was detectable in either other myeloid leukemia lines, fresh leukemic samples, or normal human myeloid cells by Northern analyses. Id1 mRNA was not detected by polymerase chain reaction in either leukemic or normal myeloid cells except in K562 myeloid/erythroid cells. These results showed that Id2 mRNA was constitutively expressed in more mature myeloid blast cells and level markedly increased with terminal myeloid differentiation, suggesting that Id2 protein may inhibit an HLH transcriptional complex that normally represses myeloid differentiation.

Acute Disease↗