Genomic and proteomic analysis of the myeloid differentiation program.

Although the mature neutrophil is one of the better characterized mammalian cell types, the mechanisms of myeloid differentiation are incompletely understood at the molecular level. A mouse promyelocytic cell line (MPRO), derived from murine bone marrow cells and arrested developmentally by a dominant-negative retinoic acid receptor, morphologically differentiates to mature neutrophils in the presence of 10 microM retinoic acid. An extensive catalog was prepared of the gene expression changes that occur during morphologic maturation. To do this, 3'-end differential display, oligonucleotide chip array hybridization, and 2-dimensional protein electrophoresis were used. A large number of genes whose mRNA levels are modulated during differentiation of MPRO cells were identified. The results suggest the involvement of several transcription regulatory factors not previously implicated in this process, but they also emphasize the importance of events other than the production of new transcription factors. Furthermore, gene expression patterns were compared at the level of mRNA and protein, and the correlation between 2 parameters was studied. (Blood. 2001;98:513-524)

Comparison of TP53 mutations identified by oligonucleotide microarray and conventional DNA sequence analysis.

As the rate of gene discovery accelerates, more efficient methods are needed to analyze genes in human tissues. To assess the efficiency, sensitivity, and specificity of different methods, alterations of TP53 were independently evaluated in 108 ovarian tumors by conventional DNA sequence analysis and oligonucleotide microarray (p53 GeneChip). All mutations identified by oligonucleotide microarray and all disagreements with conventional gel-based DNA sequence analysis were confirmed by re-analysis with manual and automated dideoxy DNA sequencing. A total of 77 ovarian cancers were identified as having TP53 mutations by one of the two approaches, 71 by microarray and 63 by gel-based DNA sequence analysis. The same mutation was identified in 57 ovarian cancers, and the same wild type TP53 sequence was observed in 31 ovarian cancers by both methods, for a concordance rate of 81%. Among the mutation analyses discordant by these methods for TP53 sequence were 14 cases identified as mutated by microarray but not by conventional DNA sequence analysis and 6 cases identified as mutated by conventional DNA sequence analysis but not by microarray. Overall, the oligonucleotide microarray demonstrated a 94% accuracy rate, a 92% sensitivity, and an 100% specificity. Conventional DNA sequence analysis demonstrated an 87% accuracy rate, 82% sensitivity, and a 100% specificity. Patients with TP53 mutations had significantly shorter overall survival than those with no mutation (P = 0.02). Women with mutations in loop2, loop3, or the loop-sheet-helix domain had shorter survival than women with other mutations or women with no mutations (P = 0.01). Although further refinement would be helpful to improve the detection of certain types of TPS3 alterations, oligonucleotide microarrays were shown to be a powerful and effective tool for TP53 mutation detection.

Sulindac suppresses tumorigenesis in the Min mouse.

The Min mouse provides a genetically defined model for inherited and sporadic forms of human colorectal tumorigenesis. To test the suitability of this model for the evaluation and optimization of chemopreventive agents, we examined the effects of sulindac on tumorigenesis in Min mice as this compound can inhibit colorectal tumorigenesis in human familial adenomatous polyposis patients. Treatment of Min mice with sulindac in their drinking water (84 mg/l) or diet (167 and 334 p.p.m.) resulted in a significantly decreased average tumor load. The conservation of sulindac activity in the Min mouse provides an opportunity to explore the mechanism of sulindac suppression as well as to test other potential chemopreventive agents.

Suppression of carcinogenesis in the intestines of min mice by the soybean-derived Bowman-Birk inhibitor.

We have performed experiments to determine whether the soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), has the ability to affect intestinal carcinogenesis in Min mice. Min mice have an autosomally dominantly inherited predisposition to multiple intestinal neoplasms and are known to have a very high spontaneous rate of tumor development in both the small intestine and colon. BBI was administered in the diet as BBI Concentrate (BBIC), the form of BBI which is currently being evaluated in human trials as a cancer chemopreventive agent. We observed that 0.5% dietary BBIC led to a 42-50% reduction in the number of tumors/mouse in the small intestine and colon and a 41% reduction of tumorigenesis in the colon when the data are analyzed in terms of the fraction of mice bearing tumors. Thus, tumor-development in both the small intestine and colon of Min mice can be significantly suppressed by BBIC, despite the fact that the animals carry a predisposing mutation that leads to a markedly increased intestinal tumor incidence and mortality rate.

Inactivation of both APC alleles in human and mouse tumors.

Germline mutations of the adenomatous polyposis coli (APC) gene lead to multiple intestinal tumors in familial adenomatous polyposis patients and in multiple intestinal neoplasia (Min) mice. Current models predict that inactivation of the remaining normal allele of a tumor suppressor gene is rate limiting for tumor formation, but this has been difficult to prove. While examination of colorectal adenomas from familial adenomatous polyposis patients identified somatic inactivating mutations of the second allele in the majority of tumors (19 of 24), the absolute requirement for an early inactivating event could not be demonstrated. In contrast, inactivation of the remaining allele of the murine APC (Apc) could be demonstrated in 100% (30 of 30) of tumors from Min mice. Moreover, inactivation was observed in the earliest recognizable phase of tumors, including some lesions containing as few as two dysplastic crypts. These results suggest that the mutation of the second APC allele is an early event in Min and familial adenomatous polyposis tumorigenesis, supporting Knudson's hypothesis.

APC mutations occur early during colorectal tumorigenesis.

Human tumorigenesis is associated with the accumulation of mutations both in oncogenes and in tumour suppressor genes. But in no common adult cancer have the mutations that are critical in the early stages of the tumorigenic process been defined. We have attempted to determine if mutations of the APC gene play such a role in human colorectal tumours, which evolve from small benign tumours (adenomas) to larger malignant tumours (carcinomas) over the course of several decades. Here we report that sequence analysis of 41 colorectal tumours revealed that the majority of colorectal carcinomas (60%) and adenomas (63%) contained a mutated APC gene. Furthermore, the APC gene met two criteria of importance for tumour initiation. First, mutations of this gene were found in the earliest tumours that could be analysed, including adenomas as small as 0.5 cm in diameter. Second, the frequency of such mutations remained constant as tumours progressed from benign to malignant stages. These data provide strong evidence that mutations of the APC gene play a major role in the early development of colorectal neoplasms.