Roberto A. Macina

Nox1 is over‐expressed in human colon cancers and correlates with activating mutations in K‐Ras

The NADPH‐oxidase 1 (Nox1) is a homolog of gp91phox, the catalytic subunit of the phagocyte superoxide‐generating NADPH‐oxidase. Nox1 is expressed in normal colon epithelial cells and in colon tumor cell lines, and overexpression in model cells has been implicated in stimulation of mitogenesis and angiogenesis and inhibition of apoptosis. This suggests that aberrant expression of Nox1 could contribute to the development of colorectal cancer. Herein, we examine the expression of Nox1 mRNA in 24 colon tumors of various stages compared with paired adjacent normal tissue from the same patient, and correlate expression with some common mutations associated with colon cancer. Nox1 was overexpressed compared with paired normal tissue in 57% of tumors as early as the adenoma stage, with no correlation of expression level with tumor stage. Overexpression of Nox1 mRNA correlated with Nox1 protein levels assessed by immunofluorescence and immunohistochemistry with an antibody specific for Nox1. There was a strong correlation between Nox1 mRNA level and activating mutations in codons 12 and 13 of K‐Ras. Eighty percent (8/10) of tumors with codons 12 and 13 mutations had a 2‐fold or more increase in Nox1 mRNA, and 70% (7/10) had a 5‐fold or greater increase. Transgenic mice expressing K‐RasG12V in the intestinal epithelium also expressed markedly elevated Nox1 in both small and large intestine. There was no correlation between inactivating mutations in the tumor suppressor p53 and Nox1 expression. We conclude that Nox1 mRNA and protein are overexpressed in colon cancer and are strongly correlated with activating mutations in K‐Ras.

Chromosomal localization of seven cloned antigen genes provides evidence of diploidy and further demonstration of karyotype variability in Trypanosoma cruzi

The karyotype of Trypanosoma cruzi was studied by pulsed field gel electrophoresis (PFGE) in conditions that allowed 20-25 chromosome bands to be detected. However, several of these bands were present in non-equimolar amounts, suggesting that the total chromosome number is considerably higher. The patterns obtained with the different cloned and uncloned strains were unique, suggesting that the karyotype of T. cruzi is highly variable. The chromosomal localizations of seven cloned genes were determined by Southern blotting of PFGE-separated chromosomes. Three of the clones gave rise to similar patterns and mapped on a chromosome or a family of chromosomes larger than 1.6 Mb. Two clones mapped on either single or pairs of chromosomes, which in some cases differed considerably in size between the different strains tested, suggesting that extensive chromosome rearrangements occur in T. cruzi. Another clone hybridized to several chromosomes in most strains and probably represents a family of genes. Lastly, one clone hybridized to nearly all chromosomes. Many of the clones hybridized to pairs of restriction fragments in the different strains, suggesting that they are allelic. For one of the clones it was possible to provide further evidence for the allelic nature of the fragments by establishing detailed restriction maps around them and by showing that the two fragments in a pair hybridized to chromosomes which differed slightly in size. Taken together, the results infer that the genome of T. cruzi epimastigotes is diploid.

Polymorphisms within minicircle sequence classes in the kinetoplast DNA of Trypanosoma cruzi clones

Four minicircle classes were analyzed using cloned minicircles as probes and single-cell cloned Trypanosoma cruzi parasites. The hybridization conditions used allowed identification of minicircle classes within kinetoplast DNA that were non-homologous to each other. Two of these minicircle classes, detected with probes pTckAWP-2 and -3, were present together in several of the CA 1 and Miranda clones, in spite of the fact that either pTckAWP-2 or both minicircle classes were undetectable in other isolates and clones of the parasite. The other two minicircle classes (pTckM-84 and -88) were located in some Miranda cloned parasites which were characterized by the simple restriction endonuclease pattern of their minicircles. Both pTckM-84 and -88 minicircle classes represented 52-71% of the kinetoplast DNA in the latter group of trypanosomes. Restriction endonuclease mapping allowed the identification of polymorphic minicircles in two of the four minicircle classes analyzed (pTckAWP-2 and pTckM-88). The polymorphisms were observed in part of the molecules of one minicircle class within a single trypanosome clone, as well as when different clones or even some of those obtained from the same isolate were compared. In addition, a different proportion of pTckM-88 type of minicircle sequence class was observed in the kinetoplast DNA from two of the Miranda clones analyzed. These observations demonstrated that similar molecules may evolve independently in sequence in each parasite. The polymorphic minicircles detected may arise from sequence variations before expansion of a future homogeneous minicircle sequence class.