Bora E. Baysal

Revisiting the TCA cycle: signaling to tumor formation

A role for mitochondria in tumor formation is suggested by mutations in enzymes of the TCA cycle: isocitrate dehydrogenase (IDH), succinate dehydrogenase (SDH) and fumarate hydratase (FH). Although they are all components of the TCA cycle, the resulting clinical presentations do not overlap. Activation of the hypoxia pathway can explain SDH phenotypes, but recent data suggest that FH and IDH mutations lead to tumor formation by repressing cellular differentiation. In this review, we discuss recent findings in the context of both mitochondrial and cytoplasmic components of the TCA cycle, and we propose that extrametabolic roles of TCA cycle metabolites result in reduced cellular differentiation. Furthermore, activation of the pseudohypoxia pathway likely promotes the growth of these neoplasias into tumors.

Centrifugal enhancement of retroviral mediated gene transfer

Centrifugation has been used for many years to enhance infection of cultured cells with a variety of different types of viruses, but it has only recently been demonstrated to be effective for retroviruses (Ho et al. (1993) J. Leukocyte Biol. 53, 208-212; Kotani et al. (1994) Hum. Gene Ther. 5, 19-28). Centrifugation was investigated as a means of increasing the transduction of a retroviral vector for gene transfer into cells with the potential for transplantation and engraftment in human patients suffering from genetic disease, i.e., gene therapy. It was found that centrifugation significantly increased the rate of transduction into adherent murine fibroblasts and into non-adherent human hematopoietic cells, including primary CD34+ enriched cells. The latter samples include cells capable of reconstitution of hematopoiesis in myeloablated patients. As a step toward optimization of this method, it was shown that effective transduction is: (1) achieved at room temperature; (2) directly related to time of centrifugation and to relative centrifugal force up to 10,000 g; (3) independent of volume of supernatant for volumes > or = 0.5 ml using non-adherent cell targets in test tubes, but dependent upon volume for coverage of adherent cell targets in flat bottom plates; and (4) inversely related to cell numbers per tube using non-adherent cells. The results support the proposal that centrifugation increases the reversible binding of virus to the cells, and together with results reported by Hodgkin et al. (Hodgkin et al. (1988) J. Virol. Methods 22, 215-230), these data support a model in which the centrifugal field counteracts forces of diffusion which lead to dissociation during the reversible phase of binding.

Nearly all hereditary paragangliomas in The Netherlands are caused by two founder mutations in the SDHD gene

Hereditary paragangliomas or glomus tumors are usually benign slow‐growing tumors in the head and neck region. The inheritance pattern of hereditary paraganglioma is autosomal dominant with imprinting. Recently, we have identified the SDHD gene encoding subunit D of the mitochondrial respiratory chain complex II as one of the genes involved in hereditary paragangliomas. Here, we demonstrate that two founder mutations, Asp92Tyr and Leu139Pro, are responsible for paragangliomas in 24 and 6 of the 32 independently ascertained Dutch paraganglioma families, respectively. These two mutations were also detected among 20 of 55 isolated patients. Ten of the isolated patients had multiple paragangliomas, and in eight of these SDHD germline mutations were found, indicating that multicentricity is a strong predictive factor for the hereditary nature of the disorder in isolated patients. In addition, we demonstrate that the maternally derived wild‐type SDHD allele is lost in tumors from mutation‐carrying patients, indicating that SDHD functions as a tumor suppressor gene.

Phenotypic dichotomy in mitochondrial complex II genetic disorders

This review presents our current knowledge on the genetic and phenotypic aspects of mitochondrial complex II gene defects. The mutations of the complex II subunits cause two strikingly different group of disorders, revealing a phenotypic dichotomy. Genetic disorders of the mitochondrial respiratory chain are often characterized by hypotonia, growth retardation, cardiomyopathy, myopathy, neuropathy, organ failure, and metabolic derangement. These disorders are transmitted through maternal lineage if the defective gene is located in the mitochondrial genome or may follow a Mendelian pattern if it is in the nucleus. Mitochondrial complex II (succinate:ubiquinone oxidoreductase) is the smallest complex in the respiratory chain and is composed of four subunits encoded by nuclear genes SDHA, SDHB, SDHC, and SDHD. Complex II oxidizes succinate to fumarate in the Krebs cycle and is involved in the mitochondrial electron transport chain. SDHA and SDHB encode the flavoprotein and iron-sulfur proteins, respectively, and SDHC and SDHD encode the two hydrophobic membrane-spanning subunits. While mutations in SDHA display a phenotype resembling other mitochondrial and Krebs cycle gene defects, those in SDHB, SDHC and SDHD cause hereditary paraganglioma. Paraganglioma is characterized by slow-growing vascular tumors of the paraganglionic tissue (i.e., adrenal and extra-adrenal paragangliomas, including those in the head and neck, mediastinum, abdomen, and pheochromocytomas). Paraganglioma caused by SDHD mutations occurs exclusively after paternal transmission, suggesting that genomic imprinting influences gene expression. Association of a mitochondrial gene defect with tumorigenesis expands the phenotypic spectrum of mitochondrial diseases and adds genomic imprinting as a new transmission mode in mitochondrial genetics. The phenotypic features of complex II gene mutations suggest that whereas the catalytic subunit SDHA mutations may compromise the Krebs cycle, those in other structural subunits may affect oxygen sensing and signaling.

Novel mutations in the SDHD gene in pedigrees with familial carotid body paraganglioma and sensorineural hearing loss

Paraganglioma (PGL) is a rare disorder characterized by tumors of the head and neck region. Between 10% and 50% of cases of PGL are familial, and the disease is autosomal dominant and subject to age‐dependent penetrance and imprinting. The paraganglioma gene (PGL1) has been mapped to 11q22.3–q23, and recently germline mutations in the SDHD gene have been identified. The SDHD region contains another gene, DPP2/TIMM8B, the homolog of which causes dystonia and deafness seen in Mohr‐Tranebjaerg syndrome. Using four PGL pedigrees, two of which exhibit coinheritance of PGL and sensorineural hearing loss or tinnitus, analysis of 14 microsatellite markers provided support for linkage to the PGL1 locus. Sequence analysis identified novel mutations in exon 1 and exon 3 of the SDHD gene, including a novel two base pair deletion in exon 3 creating a premature stop codon at position 67; a novel three base pair deletion in exon 3 resulting in the loss of Tyr‐93; a missense mutation in exon 3 resulting in the substitution of Leu‐81 for Pro‐81; and a novel G‐to‐C substitution in exon 1 resulting in the substitution of Met‐1 for Ile‐1. No base changes were detected in the DPP2/TIMM8B gene. There was no apparent loss of heterozygosity at the site of the SDHD mutations. However, RT‐PCR analysis of tumor samples showed monoallelic expression of the mutant (paternal) allele as expected for imprinting. This has not previously been shown for this disorder. The inheritance and expression of the SDHD gene is consistent with the PGL1 gene being subject to genomic imprinting.

Loss of distal 11q is associated with DNA repair deficiency and reduced sensitivity to ionizing radiation in head and neck squamous cell carcinoma

About 45% of head and neck squamous cell carcinomas (HNSCC) are characterized by amplification of chromosomal band 11q13. This amplification occurs by a breakage‐fusion‐bridge (BFB) cycle mechanism. The first step in the BFB cycle involves breakage and loss of distal 11q, from FRA11F (11q14.2) to 11qter. Consequently, numerous genes, including three critical genes involved in the DNA damage response pathway, MRE11A, ATM, and H2AFX are lost in the step preceding 11q13 amplification. We hypothesized that this partial loss of genes on distal 11q may lead to a diminished DNA damage response in HNSCC. Characterization of HNSCC using fluorescence in situ hybridization (FISH) revealed concurrent partial loss of MRE11A, ATM, and H2AFX in all four cell lines with 11q13 amplification and in four of seven cell lines without 11q13 amplification. Quantitative microsatellite analysis and loss of heterozygosity studies confirmed the distal 11q loss. FISH evaluation of a small series of HNSCC, ovarian, and breast cancers confirmed the presence of 11q loss in at least 60% of these tumors. All cell lines with distal 11q loss exhibited a diminished DNA damage response, as measured by a decrease in the size and number of γ‐H2AX foci and increased chromosomal instability following treatment with ionizing radiation. In conclusion, loss of distal 11q results in a defective DNA damage response in HNSCC. Distal 11q loss was also unexpectedly associated with reduced sensitivity to ionizing radiation. Although the literature attributes the poor prognosis in HNSCC to 11q13 gene amplification, our results suggest that distal 11q deletions may be an equally significant factor.

Genomic imprinting and environment in hereditary paraganglioma.

Hereditary paraganglioma (PGL) is characterized by the development of slow‐growing and vascularized tumors in the paraganglionic system. PGL is caused by germ line heterozygous inactivating mutations in the SDHB (PGL4), SDHC (PGL3), or SDHD (PGL1) genes, which encode three of the four subunits of mitochondrial complex II (succinate dehydrogenase; SDH). Common tumor sites include the carotid body in the neck and paraganglia in the abdomen. The risk of tumor development associated with SDHD mutations is determined by the sex of the transmitting parent, because only a paternal transmission leads to tumorigenesis in the progeny. This transmission pattern suggests operation of genomic imprinting on the SDHD gene. There is also evidence that the risk of tumor development increases at higher altitudes among SDHD mutation carriers. Accordingly, the increased prevalence of SDHD mutations in the Netherlands, attributable to multiple founder mutations, has been explained in part by the low altitudes in this country, which presumably reduce gene penetrance and relax the natural selection. Thus, PGL caused by SDHD mutations represents an unusual example of an inherited monogenic tumor syndrome because the risk of tumorigenesis shows an absolute dependence on the sex of the transmitting parent and may be modified by a ubiquitous environmental factor.

Relationship Between ERCC1 Polymorphisms, Disease Progression, and Survival in the Gynecologic Oncology Group Phase III Trial of Intraperitoneal Versus Intravenous Cisplatin and Paclitaxel for Stage III Epithelial Ovarian Cancer

PURPOSE We hypothesized that common polymorphisms in excision repair cross-complementation group 1 (ERCC1), involved in nucleotide excision repair of platinum-induced damage, would be associated with progression-free survival (PFS) and overall survival (OS) in women with optimally resected, stage III epithelial ovarian cancer (EOC) treated with cisplatin and paclitaxel (C+P). PATIENTS AND METHODS Single nucleotide polymorphism analysis was carried out by direct pyrosequencing at two sites (codon 118 and C8092A) in ERCC1 in leukocyte DNA from women who participated in the Gynecologic Oncology Group (GOG) phase III protocol-172 and were randomly assigned to intraperitoneal or intravenous C+P. RESULTS ERCC1 genotyping was performed in 233 of the 429 women who participated in GOG-172. The genotype distribution at codon 118 was 17% with C/C, 43% with C/T, and 40% with T/T, and the genotype distribution at C8092A was 56% with C/C, 37% with C/A, and 7% with A/A. Adjusted Cox regression analysis revealed that the codon 118 polymorphism in ERCC1 was not significantly associated with disease progression or death. Women with the C8092A C/A or A/A genotypes compared with the C/C genotype had an increased risk of disease progression (hazard ratio [HR] = 1.44; 95% CI, 1.06 to 1.94; P = .018) and death (HR = 1.50; 95% CI, 1.07 to 2.09; P = .018). Median PFS and OS were 6 and 17 months shorter for women with the C8092A C/A or A/A genotypes versus the C/C genotype, respectively. CONCLUSION Although the ERCC1 codon 118 polymorphism does not seem to be associated with clinical outcome, the C8092A polymorphism was an independent predictor of PFS and OS in women with optimally resected EOC.

APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages

The extent, regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific C>U RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins, including many that are involved in pathogenesis of viral diseases. APOBEC3A, which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons, mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of C>U RNA editing in mammals.

Biochemically silent abdominal paragangliomas in patients with mutations in the succinate dehydrogenase subunit B gene.

CONTEXT Patients with adrenal and extra-adrenal abdominal paraganglioma (PGL) almost invariably have increased plasma and urine concentrations of metanephrines, the O-methylated metabolites of catecholamines. We report four cases of biochemically silent abdominal PGL, in which metanephrines were normal despite extensive disease. OBJECTIVE Our objective was to identify the mechanism underlying the lack of catecholamine hypersecretion and metabolism to metanephrines in biochemically silent PGL. DESIGN This is a descriptive study. SETTING The study was performed at a referral center. PATIENTS One index case and three additional patients with large abdominal PGL and metastases but with the lack of evidence of catecholamine production, six patients with metastatic catecholamine-producing PGL and a mutation of the succinate dehydrogenase subunit B (SDHB) gene, and 136 random patients with catecholamine-producing PGL were included in the study. MAIN OUTCOME MEASURES Plasma, urine, and tumor tissue concentrations of catecholamines and metabolites were calculated with electron microscopy and tyrosine hydroxylase immunohistochemistry. RESULTS All four patients with biochemically silent PGL had an underlying SDHB mutation. In the index case, the tumor tissue concentration of catecholamines (1.8 nmol/g) was less than 0.01% that of the median (20,410 nmol/g) for the 136 patients with catecholamine-producing tumors. Electron microscopy showed the presence of normal secretory granules in all four biochemically silent PGLs. Tyrosine hydroxylase immunoreactivity was negligible in the four biochemically silent PGLs but abundant in catecholamine-producing PGLs. CONCLUSIONS Patients with SDHB mutations may present with biochemically silent abdominal PGLs due to defective catecholamine synthesis resulting from the absence of tyrosine hydroxylase. Screening for tumors in patients with SDHB mutations should not be limited to biochemical tests of catecholamine excess.