Olga Chernova

The p53 Network

Loss of control of genomic stability is central in the development of cancer, and p53, by regulating normal responses to DNA damage and other forms of genotoxic stress, is a key element in maintaining genomic stability. Thus, it is no surprise that functional p53 is lost in about half of all human cancers. What about the other half? One possibility is that p53-independent regulatory mechanisms have been lost. Another is that inactivation of p53-dependent pathways can occur at any of several different points and that p53 itself is merely the most common target. For example, the p53 inhibitor Mdm2 is overexpressed in tumors independently of the p53 mutation. Here, we review pathways that signal in to p53, in response to different forms of stress, and pathways that signal out, triggered by activated p53. It is clear that p53 is the central component of a complex network of signaling pathways and that the other components of these pathways pose alternative targets for inactivation. For additional recent reviews, see Refs. 1 and 2.

The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis

Gliomas are the most common primary tumours of the central nervous system, with nearly 15,000 diagnosed annually in the United States and a lethality approaching 80% within the first year of glioblastoma diagnosis. The marked induction of angiogenesis in glioblastomas suggests that it is a necessary part of malignant progression; however, the precise molecular mechanisms underlying the regulation of brain tumour growth and angiogenesis remain unresolved. Here we report that a candidate tumour suppressor gene, ING4, is involved in regulating brain tumour growth and angiogenesis. Expression of ING4 is significantly reduced in gliomas as compared with normal human brain tissue, and the extent of reduction correlates with the progression from lower to higher grades of tumours. In mice, xenografts of human glioblastoma U87MG, which has decreased expression of ING4, grow significantly faster and have higher vascular volume fractions than control tumours. We show that ING4 physically interacts with p65 (RelA) subunit of nuclear factor NF-κB, and that ING4 regulates brain tumour angiogenesis through transcriptional repression of NF-κB-responsive genes. These results indicate that ING4 has an important role in brain tumour pathogenesis.

Inhibition of constitutively active Stat3 suppresses proliferation and induces apoptosis in glioblastoma multiforme cells

Glioblastoma multiforme (GBM), the most common and malignant central nervous system tumor in humans, is highly proliferative and resistant to apoptosis. Stat3, a latent transcription factor being activated by aberrant cytokine or growth factor signaling, acts as a suppressor of apoptosis in a number of cancer cells. Here we report that GBM tumors and cell lines contain high levels of constitutively activated Stat3 when compared with normal human astrocytes, white matter, and normal tissue adjacent to tumor. The persistent activation of Stat3 is in part, attributable to an autocrine action of interleukin-6 in the GBM cell line U251. Janus kinase inhibitor AG490 inhibits Stat3 activation with a concomitant reduction in steady-state levels of Bcl-XL, Bcl-2 and Mcl-1 proteins and induces apoptosis in U251 cells as revealed by Poly (ADP-ribose) polymerase cleavage and Annexin-V staining. Expression of a dominant negative mutant Stat3 protein or treatment with AG490 markedly reduces the proliferation of U251 cells by inhibiting the constitutive activation of Stat3. These results provide evidence that constitutive activation of Stat3 contributes to the pathogenesis of glioblastoma by promoting both proliferation and survival of GBM cells. Therefore, targeting Stat3 signaling may provide a potential therapeutic intervention for GBM.

A novel gene, LGI1 , from 10q24 is rearranged and downregulated in malignant brain tumors

Loss of heterozygosity for 10q23–26 is seen in over 80% of glioblastoma multiforme tumors. We have used a positional cloning strategy to isolate a novel gene, LGI1 (Leucine-rich gene–Glioma Inactivated), which is rearranged as a result of the t(10;19)(q24;q13) balanced translocation in the T98G glioblastoma cell line lacking any normal chromosome 10. Rearrangement of the LGI1 gene was also detected in the A172 glioblastoma cell line and several glioblastoma tumors. These rearrangements lead to a complete absence of LGI1 expression in glioblastoma cells. The LGI1 gene encodes a protein with a calculated molecular mass of 60 kD and contains 3.5 leucine-rich repeats (LRR) with conserved flanking sequences. In the LRR domain, LGI1 has the highest homology with a number of transmembrane and extracellular proteins which function as receptors and adhesion proteins. LGI1 is predominantly expressed in neural tissues, especially in brain; its expression is reduced in low grade brain tumors and it is significantly reduced or absent in malignant gliomas. Its localization to the 10q24 region, and rearrangements or inactivation in malignant brain tumors, suggest that LGI1 is a candidate tumor suppressor gene involved in progression of glial tumors.

The role of p53 in regulating genomic stability when DNA and RNA synthesis are inhibited

In addition to its induction by DNA damage, p53 is induced by drugs that starve cells for DNA and RNA precursors, or by inhibitors of DNA or RNA polymerase. In normal cells, the induction of p53 by dNTP starvation serves a protective role, mediating rapid, reversible cell-cycle arrest without DNA damage. In most cell lines, this first line of defense is missing, so that starvation for dNTPs causes DNA to break, thus increasing the probability of genomic instability, chromosome deletions and gene amplification. The mechanism of how p53 is induced remains unclear.

MYC Abrogates p53-Mediated Cell Cycle Arrest in N-(Phosphonacetyl)-L-Aspartate-Treated Cells, Permitting CAD Gene Amplification

ABSTRACT Genomic instability, including the ability to undergo gene amplification, is a hallmark of neoplastic cells. Similar to normal cells, “nonpermissive” REF52 cells do not develop resistance toN-(phosphonacetyl)-l-aspartate (PALA), an inhibitor of the synthesis of pyrimidine nucleotides, through amplification of cad, the target gene, but instead undergo protective, long-term, p53-dependent cell cycle arrest. Expression of exogenous MYC prevents this arrest and allows REF52 cells to proceed to mitosis when pyrimidine nucleotides are limiting. This results in DNA breaks, leading to cell death and, rarely, to cad gene amplification and PALA resistance. Pretreatment of REF52 cells with a low concentration of PALA, which slows DNA replication but does not trigger cell cycle arrest, followed by exposure to a high, selective concentration of PALA, promotes the formation of PALA-resistant cells in which the physically linked cad and endogenous N-myc genes are coamplified. The activated expression of endogenous N-myc in these pretreated PALA-resistant cells allows them to bypass the p53-mediated arrest that is characteristic of untreated REF52 cells. Our data demonstrate that two distinct events are required to form PALA-resistant REF52 cells: amplification ofcad, whose product overcomes the action of the drug, and increased expression of N-myc, whose product overcomes the PALA-induced cell cycle block. These paired events occur at a detectable frequency only when the genes are physically linked, ascad and N-myc are. In untreated REF52 cells overexpressing N-MYC, the level of p53 is significantly elevated but there is no induction of p21 waf1 expression or growth arrest. However, after DNA is damaged, the activated p53 executes rapid apoptosis in these REF52/N-myc cells instead of the long-term protective arrest seen in REF52 cells. The predominantly cytoplasmic localization of stabilized p53 in REF52/N-myc cells suggests that cytoplasmic retention may help to inactivate the growth-suppressing function of p53.

Molecular organization of internal telomeric sequences in Chinese hamster chromosomes

In Chinese hamster extended blocks of telomeric-like repeats were previously detected by in situ hybridization at the pericentromeric region of most chromosomes and short arrays were localized at several interstitial sites. In this work, we analyzed the molecular organization of internal telomeric sequences (ITs) in the Chinese hamster genome. In genomic transfers hybridized with a telomeric probe, multiple Bal31 insensitive fragments were detected. Most of the fragments ranged in size between less than 1 kb and more than 100 kb and some were polymorphic. Fluorescence in situ hybridization experiments on DNA fibers and on elongated chromosomes showed that the pericentromeric ITs are composed of extensive and essentially continuous arrays of telomeric-like sequences. We then isolated three genomic regions which contain short ITs. These ITs are localized at interstitial sites (3q13-15, 3q21-26, 1p26) and are composed of 29-126 bp of (TTAGGG)(n) repeats. A peculiar feature of all the three ITs is the AT richness of the flanking sequences. Since AT-rich DNA is known to be unstable and characteristic of several mammalian fragile sites, we propose that the three ITs were inserted at these sites during the repair of double strand breaks.

A novel member of the WD-repeat gene family, WDR11, maps to the 10q26 region and is disrupted by a chromosome translocation in human glioblastoma cells.

Allelic deletions of 10q25–26 and 19q13.3–13.4 are the most common genetic alterations in glial tumors. We have identified a balanced t(10;19) reciprocal translocation in the A172 glioblastoma cell line which involves both critical regions on chromosomes 10 and 19. In addition, loss of an entire copy of chromosome 10 has occurred in this cell line suggesting that the translocation event may provide a highly specific critical inactivating event in a gene responsible for tumorigenesis. Positional cloning of this translocation breakpoint resulted in the identification of a novel chromosome 10 gene, WDR11, which is a member of the WD-repeat gene family. The WDR11 gene is ubiquitously expressed, including normal brain and glial tumors. WDR11 is composed of 29 exons distributed over 58 kilobases and oriented towards the telomere. The translocation resulted in deletion of exon 5 and consequently fusion of intron 4 of WDR11 to the 3′ untranslated region of a novel member, ZNF320, of the Krüppel-like zinc finger gene family. Since ZNF320 is oriented toward the centromere of chromosome 19, both genes appeared on the same derivative chromosome der(10). The chimeric transcript encodes the WDR11 polypeptide, which is truncated after the second of six WD-repeats. ZNF320 is also expressed in A172 cells, although it is not clear if the translocation affects the expression of the altered gene because of the presence of another unrearranged gene on chromosome 19. We suggest that, because of its localization in a region frequently showing LOH and the observation of inactivation of this gene in glioblastoma cells, WDR11 is a candidate gene for the frequently proposed tumor suppressor gene in 10q25–26 which is involved in tumorigenesis of glial and other tumors showing frequent alterations in the distal 10q region.

Molecular Definition of Chromosome Translocations Involving 10q24 and 19q13 in Human Malignant Glioma Cells

Loss of heterozygosity (LOH) analysis has repeatedly implicated the 10q24-26 region as the site of tumor suppressor genes involved in the development of malignant human gliomas. However, deletions of this kind are generally too big to pinpoint the critical genes involved. On the other hand, chromosome translocations frequently interrupt genes important in the development of the phenotype. We have screened a series of cell lines and cultures from primary human brain tumors for translocations involving chromosomes 10 and 19 by using fluorescence in situ hybridization (FISH) and chromosome-specific paints. The T98G cell line carries an apparently reciprocal t(10;19)(q24;q13) translocation, the breakpoints on chromosomes 10 and 19 occurring in a region frequently showing LOH in gliomas as well as oligodendrogliomas and astrocytomas. One glioblastoma tumor, CCF 4, also showed a subtle translocation of chromosome 10 material into the long arm of chromosome 11. FISH analysis of these rearrangements showed that the chromosome 10-specific yeast artificial chromosome (YAC) 912C7 spans the translocation breakpoint in T98G cells and is also present in the material translocated from chromosome 10 in tumor CCF 4. The translocation breakpoint in 19q13 in T98G occurs within a 500-kb region of YAC 751E12. These translocation breakpoints are within the regions showing frequent LOH in brain tumors and provide a more refined tool for the identification of genes in this region involved in tumorigenesis.

Aging of mice is associated with p16(Ink4a)- and β-galactosidase-positive macrophage accumulation that can be induced in young mice by senescent cells

Senescent cells (SCs) have been considered a source of age-related chronic sterile systemic inflammation and a target for anti-aging therapies. To understand mechanisms controlling the amount of SCs, we analyzed the phenomenon of rapid clearance of human senescent fibroblasts implanted into SCID mice, which can be overcome when SCs were embedded into alginate beads preventing them from immunocyte attack. To identify putative SC killers, we analyzed the content of cell populations in lavage and capsules formed around the SC-containing beads. One of the major cell types attracted by secretory factors of SCs was a subpopulation of macrophages characterized by p16(Ink4a) gene expression and β-galactosidase activity at pH6.0 (β-galpH6), thus resembling SCs. Consistently, mice with p16(Ink4a) promoter-driven luciferase, developed bright luminescence of their peritoneal cavity within two weeks following implantation of SCs embedded in alginate beads. p16(Ink4a)/β-galpH6-expressing cells had surface biomarkers of macrophages F4/80 and were sensitive to liposomal clodronate used for the selective killing of cells capable of phagocytosis. At the same time, clodronate failed to kill bona fide SCs generated in vitro by genotoxic stress. Old mice with elevated proportion of p16(Ink4a)/β-galpH6-positive cells in their tissues demonstrated reduction of both following systemic clodronate treatment, indicating that a significant proportion of cells previously considered to be SCs are actually a subclass of macrophages. These observations point at a significant role of p16(Ink4a)/β-galpH6-positive macrophages in aging, which previously was attributed solely to SCs. They require re-interpretation of the mechanisms underlying rejuvenating effects following eradication of p16(Ink4a)/β-galpH6-positive cells and reconsideration of potential cellular target for anti-aging treatment.