Sarah F. Giardina

Association of survival and disease progression with chromosomal instability: A genomic exploration of colorectal cancer

During disease progression the cells that comprise solid malignancies undergo significant changes in gene copy number and chromosome structure. Colorectal cancer provides an excellent model to study this process. To indentify and characterize chromosomal abnormalities in colorectal cancer, we performed a statistical analysis of 299 expression and 130 SNP arrays profiled at different stages of the disease, including normal tissue, adenoma, stages 1–4 adenocarcinoma, and metastasis. We identified broad (> 1/2 chromosomal arm) and focal (< 1/2 chromosomal arm) events. Broad amplifications were noted on chromosomes 7, 8q, 13q, 20, and X and broad deletions on chromosomes 4, 8p, 14q, 15q, 17p, 18, 20p, and 22q. Focal events (gains or losses) were identified in regions containing known cancer pathway genes, such as VEGFA, MYC, MET, FGF6, FGF23, LYN, MMP9, MYBL2, AURKA, UBE2C, and PTEN. Other focal events encompassed potential new candidate tumor suppressors (losses) and oncogenes (gains), including CCDC68, CSMD1, POLR1D, and PMEPA1. From the expression data, we identified genes whose expression levels reflected their copy number changes and used this relationship to impute copy number changes to samples without accompanying SNP data. This analysis provided the statistical power to show that deletions of 8p, 4p, and 15q are associated with survival and disease progression, and that samples with simultaneous deletions in 18q, 8p, 4p, and 15q have a particularly poor prognosis. Annotation analysis reveals that the oxidative phosphorylation pathway shows a strong tendency for decreased expression in the samples characterized by poor prognosis.

Micromolar L-glutamate induces extensive apoptosis in an apoptotic-necrotic continuum of insult-dependent, excitotoxic injury in cultured cortical neurones

Excitotoxicity induced by L-glutamate (Glu), when examined in a pure neuronal cortical culture, involved widespread apoptosis at concentrations of 1-10 microM as part of a continuum of injury, which at its most servere was purely necrotic. Cells, maintained in chemically defined neurobasal/B27 medium, were exposed at d7 for 2 h to Glu (1-500 microM), and cellular injury was analysed 2 and 24 h after insult using morphology (phase-contrast microscopy), a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay, nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), terminal transferase-mediated dUTP nick end-labelling (TUNEL) and DNA fragmentation by gel electrophoresis. Glu-mediated neurotoxicity was prevented by MK-801 (5 microM), whilst CNQX (20 microM) attenuated injury by 20%. Exposure to intensive insults (100 and 500 microM Glu) induced necrosis characterized by rapid cell swelling (< 2 h) and lack of chromatin condensation, confirmed by DAPI nuclear staining. In contrast, mild insults (< 20 microM Glu) failed to produce acute neuronal swelling at < 2 h, but 24 h after injury resulted in a large number of apoptotic nuclei as confirmed by TUNEL and electrophoretic evidence of DNA fragmentation, which was attenuated by cycloheximide (0.1 microg/ml). Our findings indicate for the first time that physiological concentrations of Glu produce neuronal injury across a continuum involving apoptosis (< 20 microM) and increasingly necrosis(> 20 microM), dependent on the severity of the initial insult.

CpG Island Methylator Phenotype Associates with Low-Degree Chromosomal Abnormalities in Colorectal Cancer

Purpose: Aberrant promoter methylation and genomic instability occur frequently during colorectal cancer development. CpG island methylator phenotype (CIMP) has been shown to associate with microsatellite instability, and BRAF mutation and is often found in the right-side colon. Nevertheless, the relative importance of CIMP and chromosomal instability (CIN) for tumorigenesis has yet to be thoroughly investigated in sporadic colorectal cancers. Experimental Design: We determined CIMP in 161 primary colorectal cancers and 66 matched normal mucosae using a quantitative bisulfite/PCR/ligase detection reaction (LDR)/Universal Array assay. The validity of CIMP was confirmed in a subset of 60 primary tumors using MethyLight assay and five independent markers. In parallel, CIN was analyzed in the same study cohort using Affymetrix 50K Human Mapping arrays. Results: The identified CIMP-positive cancers correlate with microsatellite instability (P = 0.075) and the BRAF mutation V600E (P = 0.00005). The array-based high-resolution analysis of chromosomal aberrations indicated that the degree of aneuploidy is spread over a wide spectrum among analyzed colorectal cancers. Whether CIN was defined by copy number variations in selected microsatellite loci (criterion 1) or considered as a continuous variable (criterion 2), CIMP-positive samples showed a strong correlation with low-degree chromosomal aberrations (P = 0.075 and P = 0.012, respectively). Similar correlations were observed when CIMP was determined by MethyLight assay (P = 0.001 and P = 0.013, respectively). Conclusion: CIMP-positive tumors generally possess lower chromosomal aberrations, which may only be revealed using a genome-wide approach. The significant difference in the degree of chromosomal aberrations between CIMP-positive and the remainder of samples suggests that epigenetic (CIMP) and genetic (CIN) abnormalities may arise from independent molecular mechanisms of tumor progression.

Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1

Abstract: Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1‐phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)‐mediated apoptosis. Using cultures of CGCs, we found that a 24‐h exposure to KA (1–3,000 µM) induced a concentration‐dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase‐mediated nick end‐DNA labeling (TUNEL) method. KA induced a time‐ and concentration‐dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA‐induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (50 µM), indicating a KA receptor‐mediated effect. Here we present evidence for the first time that KA‐induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor‐mediated apoptosis.

Kainate-induced apoptosis correlates with c-Jun activation in cultured cerebellar granule cells

We have investigated the involvement of c‐Jun in cell death induced by exposure of primary cultures of murine cerebellar granule cells to the glutamate receptor agonist kainate (KA) and evaluated its possible use as a marker for apoptosis. Using cerebellar granule cell neurones from postnatal day 7 mice, we found that 1 hr exposure to KA (1–1000 μM) induced a concentration‐dependent neuronal cell death with characteristic apoptotic morphology, including cell shrinkage, neurite blebbing and DNA fragmentation. In addition KA‐induced a concentration‐dependent expression of c‐Jun mRNA and protein as determined by in situ hybridization and immunocytochemistry respectively. DNA fragmentation was detected using terminal transferase‐mediated nick‐end (TUNEL) labelling and agarose gel electrophoresis. KA‐induced cell death was significantly attenuated by the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; 50 μM), which shifted the concentration‐response curve significantly rightward. The number of apoptotic cell bodies, determined by TUNEL, was also reduced by CNQX (50 μM), with only 15–20% of neurones staining positive after exposure to 1mM KA. In addition, the number of positively stained cells for c‐Jun protein and mRNA was substantially reduced by CNQX (50 μM) as determined by random and representative cell counts. These results show for the first time that KA induced apoptotic neuronal death in cultured murine cerebellar granule cells involves the induction of c‐Jun mRNA and protein, suggesting the involvement of this immediate early gene in excitotoxic receptor‐mediated apoptosis and its potential use as a marker for apoptotic cell death. J. Neurosci. Res. 52:69–82, 1998.

The Signatures of Autozygosity among Patients with Colorectal Cancer

Previous studies have shown that among populations with a high rate of consanguinity, there is a significant increase in the prevalence of cancer. Single nucleotide polymorphism (SNP) array data (Affymetrix, 50K XbaI) analysis revealed long regions of homozygosity in genomic DNAs taken from tumor and matched normal tissues of colorectal cancer (CRC) patients. The presence of these regions in the genome may indicate levels of consanguinity in the individuals family lineage. We refer to these autozygous regions as identity-by-descent (IBD) segments. In this study, we compared IBD segments in 74 mostly Caucasian CRC patients (mean age of 66 years) to two control data sets: (a) 146 Caucasian individuals (mean age of 80 years) who participated in an age-related macular degeneration (AMD) study and (b) 118 cancer-free Caucasian individuals from the Framingham Heart Study (mean age of 67 years). Our results show that the percentage of CRC patients with IBD segments (>or=4 Mb length and 50 SNPs probed) in the genome is at least twice as high as the AMD or Framingham control groups. Also, the average length of these IBD regions in the CRC patients is more than twice the length of the two control data sets. Compared with control groups, IBD segments are found to be more common among individuals of Jewish background. We believe that these IBD segments within CRC patients are likely to harbor important CRC-related genes with low-penetrance SNPs and/or mutations, and, indeed, two recently identified CRC predisposition SNPs in the 8q24 region were confirmed to be homozygous in one particular patient carrying an IBD segment covering the region.

GROα Is Highly Expressed in Adenocarcinoma of the Colon and Down-Regulates Fibulin-1

Purpose: The growth-related oncogene α (GROα) is a secreted interleukin-like molecule that interacts with the CXCR2 G-protein–coupled receptor. We found that the mRNA and protein products of GROα are more highly expressed in neoplastic than normal colon epithelium, and we studied potential mechanisms by which GROα may contribute to tumor initiation or growth. Experimental Design: Cell lines that constitutively overexpress GROα were tested for growth rate, focus formation, and tumor formation in a xenograft model. GROα expression was determined by Affymetrix GeneChip (241 microdissected colon samples), real-time PCR (n = 32), and immunohistochemistry. Primary colon cancer samples were also employed to determine copy number changes and loss of heterozygosity related to the GROα and fibulin-1 genes. Results: In cell cultures, GROα transfection transformed NIH 3T3 cells, whereas inhibition of GROα by inhibitory RNA was associated with apoptosis, decreased growth rate, and marked up-regulation of the matrix protein fibulin-1. Forced expression of GROα was associated with decreased expression of fibulin-1. Expression of GROα mRNA was higher in primary adenocarcinomas (n = 132), adenomas (n = 32), and metastases (n = 52) than in normal colon epithelium (P < 0.001). These results were confirmed by real-time PCR and by immunohistochemistry. Samples of primary and metastatic colon cancer showed underexpression of fibulin-1 when compared with normal samples. There were no consistent changes in gene copy number of GROα or fibulin-1, implying a transcriptional basis for these findings. Conclusion: Elevated expression of GROα is frequent in adenocarcinoma of the colon and is associated with down-regulation of the matrix protein fibulin-1 in experimental models and in clinical samples. GROα overexpression abrogates contact inhibition in cell culture models, whereas inhibition of GROα expression is associated with apoptosis. Importantly, coexpression of fibulin-1 with GROα abrogates key aspects of the transformed phenotype, including tumor formation in a murine xenograft model. Targeting GRO proteins may provide new opportunities for treatment of colon cancer.

Emerging Paradigms in Cancer Genetics: Some Important Findings from High-Density Single Nucleotide Polymorphism Array Studies

High-density single nucleotide polymorphism (SNP) mapping arrays have identified chromosomal features whose importance to cancer predisposition and progression is not yet clearly defined. Of interest is that the genomes of normal somatic cells (reflecting the combined parental germ-line contributions) often contain long homozygous stretches. These chromosomal segments may be explained by the common ancestry of the individuals parents and thus may also be called autozygous. Several studies link consanguinity to higher rates of cancer, suggesting that autozygosity (a genomic consequence of consanguinity) may be a factor in cancer predisposition. SNP array analysis has also identified chromosomal regions of somatic uniparental disomy (UPD) in cancer genomes. These are chromosomal segments characterized by loss of heterozygosity (LOH) and a normal copy number (two) but which are not autozygous in the germ-line or normal somatic cell genome. In this review, we will also discuss a model [cancer gene activity model (CGAM)] that may explain how autozygosity influences cancer predisposition. CGAM can also explain how the occurrence of certain chromosomal aberrations (copy number gain, LOH, and somatic UPDs) during carcinogenesis may be dependent on the germ-line genotypes of important cancer-related genes (oncogenes and tumor suppressors) found in those chromosomal regions.

Incorporation of sodium channel blocking and free radical scavenging activities into a single drug, AM-36, results in profound inhibition of neuronal apoptosis

AM‐36 is a novel neuroprotective agent incorporating both antioxidant and Na+ channel blocking actions. In cerebral ischaemia, loss of cellular ion homeostasis due to Na+ channel activation, together with increased reactive oxygen species (ROS) production, are thought to contribute to neuronal death. Since neuronal death in the penumbra of the ischaemic lesion is suggested to occur by apoptosis, we investigated the ability of AM‐36, antioxidants and Na+ channel antagonists to inhibit toxicity induced by the neurotoxin, veratridine in cultured cerebellar granule cells (CGCs). Veratridine (10 – 300 μM) concentration‐dependently reduced cell viability of cultured CGCs. Under the experimental conditions employed, cell death induced by veratridine (100 μM) possessed the characteristics of apoptosis as assessed by morphology, TUNEL staining and DNA laddering on agarose gels. Neurotoxicity and apoptosis induced by veratridine (100 μM) were inhibited to a maximum of 50% by the antioxidants, U74500A (0.1 – 10 μM) and U83836E (0.03 – 10 μM), and to a maximum of 30% by the Na+ channel blocker, dibucaine (0.1 – 100 μM). In contrast, AM‐36 (0.01 – 10 μM) completely inhibited veratridine‐induced toxicity (IC50 1.7 (1.5 – 1.9) μM, 95% confidence intervals (CI) in parentheses) and concentration‐dependently inhibited apoptosis. These findings suggest veratridine‐induced toxicity and apoptosis are partially mediated by generation of ROS. AM‐36, which combines both Na+ channel blocking and antioxidant activity, provided superior neuroprotection compared with agents possessing only one of these actions. This bifunctional profile of activity may underlie the potent neuroprotective effects of AM‐36 recently found in a stroke model in conscious rats.

Kainate receptor-mediated apoptosis in primary cultures of cerebellar granule cells is attenuated by mitogen-activated protein and cyclin-dependent kinase inhibitors

Previous studies have suggested that neuronal apoptosis is the result of an abortive attempt to re‐enter the cell cycle, and more recently the cyclin‐dependent (CDKs) and the mitogen‐activated protein (MAP) kinases, two superfamilies of kinases that influence and control cell cycle progression, have been implicated in neuronal apoptosis. Here, to examine whether CDK/MAPK related pathways are involved in excitotoxicity, we studied the actions of various kinase inhibitors on apoptosis induced by the ionotropic glutamate (Glu) receptor agonist, kainate (KA), in primary cultures of murine cerebellar granule cells (CGCs). KA‐mediated neurotoxicity was concentration‐dependent, as determined by a cell viability assay monitoring the reduction of 3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), and largely apoptotic in nature, as shown by morphological examination and labelling of DNA fragmentation in situ using terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP digoxigenin nick‐end labelling (TUNEL). KA‐mediated neurotoxicity and apoptosis was completely attenuated by the mixed CDK and MAP kinase inhibitor, olomoucine, in a concentration‐dependent manner (50 – 600 μM), and partially by roscovitine (1 – 100 μM), a more selective CDK inihibitor. The p38 MAP kinase inhibitor, SB203580 (1 – 100 μM), partially attenuated KA receptor‐mediated apoptosis, as did the MAP kinase kinase inhibitors PD98509 (1 – 100 μM) and U0126 (1 – 100 μM). These findings provide new evidence for a complex network of interacting pathways involving CDK/MAPK that control apoptosis downstream of KA receptor activation in excitotoxic neuronal cell death.