Martina L. Veigl

DNA base modification: Ionized base pairs and mutagenesis

The nature of hydrogen bonding between normal and modified bases has been re-examined. It is proposed that hydrogen-bonding schemes may involve tautomeric, ionized or conformational forms (syn, anti and wobble). Several important cases are presented or reviewed in which physical evidence indicates the existence of ionized base pairs. When thermodynamic values determined in aqueous solution under physiological conditions are considered, it can be argued that base ionization will contribute substantially to the stability of many biologically relevant base pairs containing modified bases. A significant incidence of ionized bases in DNA may have important kinetic ramifications for the further chemical reactivity of both the modified base and its cross-strand pairing partner. Moreover, DNA structure at and surrounding ionized base pairs may be altered. For this reason, the model presented in this study should be useful as DNA-sequence analysis becomes more commonly applied to the study of mutagenesis.

Novel recurrently mutated genes in African American colon cancers

Significance Colorectal cancer is a leading cause of cancer-related deaths world-wide. African Americans exhibit the highest colon cancer incidence and mortality among all ethnic groups in the United States. Despite this finding, there is a dearth of knowledge on the genetic mechanisms underlying colon carcinogenesis in African Americans. We thus initiated this study to characterize the mutational landscapes of African American colon cancers. We identified new genes that are significantly mutated in colon cancer and that are highly preferentially targeted for mutations in colon cancers arising in African Americans as compared with Caucasians. These findings suggest differences in routes of colon carcinogenesis between the different ethnic groups and also may have implications for the ethnicity associated differences in tumor incidence and outcome. We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs. Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes. These previously unsuspected differences in the mutational landscapes of CRCs arising among individuals of different ethnicities have potential to impact on broader disparities in cancer behaviors.

INVESTIGATION OF CALMODULIN‐REGULATED NAD KINASE IN PROLIFERATING FIBROBLASTS*

The divalent cation, Ca2+, serves as a regulatory signal through which eukaryotic cells perceive and respond to both internal and external stimuli. A single protein, calmodulin (CaM), is the primary intracellular receptor for Ca2+ in most tissues where it serves as a Ca2+-dependent activator of numerous enzyme systems. Various lines of evidence suggest that the deregulation of animal cell growth upon cellular transformation involves the alteration of major cascade regulation systems, possibly those controlled by CaM. The availability of well characterized temperature-sensitive transformation-defective mutants of avian RNA tumor viruses offers an attractive system for identifying and investigating any specific role(s) which CaM plays in normal cell division and/or cellular transformation. Activation of biosynthetic pathways is required for cell proliferation. The enzyme NAD kinase is a key regulatory activity for many biosynthetic pathways since its product, NADP, is an obligatory cofactor for anabolic enzymes catalyzing oxidation/reduction reactions. NAD kinase from the pea, Pisum sativum L. has been shown by other workers to be Ca*+-CaM regulated. [Plant Physiol. 59: 55-60 (1977); Biochem. Biophys. Res. Commun. 84: 595-602 (1978)) As CaM is a highly conserved regulatory protein in both structure and function, it seemed likely that NAD kinase from animal systems is also CaM regulated. This study provides a preliminary investigation of the regulation of vertebrate NAD kinase and its potential regulation by CaM in both normal and Rous sarcoma virustransformed chick embryo fibroblasts (CEF). An NAD kinase assay was developed, based on assay conditions described by Muto and Miyachi [Plant Physiol. 59: 55-60 (1977)l. High pressure liquid chromatography (HPLC) methodology was employed to separate and quantitate the NADP produced during incubation. NAD kinase activity was found in the soluble fraction of both normal (0.5-1.4 nmoles NADP produced/hr/lO cells) and transformed fibroblasts (2-3 nmoles NADP produced/hr/lo6 cells). Transformed cells appeared to contain elevated levels of the enzyme. However a more detailed study is required to confirm this observation, since the enzyme levels in crude extracts vary among cell preparations-probably reflecting the growth rate and the distribution of cells at various stages in the cell cycle. In both normal and transformed cells the NAD kinase activity was enhanced 2-3 fold upon the addition of EGTA. Further investigation revealed that this apparent EGTA activation simply reflected a slower NADP utilization in the presence of Caz+. A time-course study, in which exogenous NADP was added to the in vitro assay mix, revealed a rapid utilization of 1.6-2.1 nmoles of the nucleotide; additional incubation resulted in no further metabolism of NADP.

Reply to Ashktorab et al.: Mutational landscape of colon cancers in African Americans

We are pleased to read the letter by Ashktorab et al. (1). We agree with Ashktorab et al. on the need for additional whole-exome sequencing of colon cancers arising in African Americans and on the value of continuing to add more cases to the numbers of African American colon cancers that have been characterized. The additional 11 African American colon cancers reported by Ashktorab et al. will be a helpful addition in this regard (2). We agree with Ashktorab et al. that the absence of mutations in EPH receptor A6 (EPHA6) and Folliculin (FLCN) from the 11 microsatellite-stable (MSS) samples they studied is not surprising, given that less than one mutation in either of these genes would be expected. In the interest of clarity, we point out that neither APC nor Kirsten rat sarcoma viral oncogene homolog (KRAS) were included in the list of new genes our study identified as significantly mutated in African American colon cancers (3). However, both our study (ref. 3, table S14) and studies by others (4) all detected a higher rate of KRAS mutations in colon cancers from African Americans compared with Caucasians. We also agree with Ashktorab et al. that WD repeat domain 87 (WDR87) is significantly mutated in colon cancers arising in African Americans, although our study found that WDR87 is also a target for mutations in colon cancers arising among Caucasians, and thus it is not in the top 15 genes whose mutations we found are highly associated with colon cancers arising in African Americans (3). We join Ashktorab et al. in hoping that the initial findings reported in PNAS will spur larger efforts in studies of ethnicity-associated differences in the mutational landscapes of colon and other cancers, as well as studies aimed at elucidating the underlying biological mechanisms by which these differences arise.