Grace L. Shen-Ong

Discovery of Novel Tumor Markers of Pancreatic Cancer using Global Gene Expression Technology

Despite several advances in our basic understanding and in the clinical management of pancreatic cancer, virtually all patients who will be diagnosed with pancreatic cancer will die from this disease. The high mortality of pancreatic cancer is predominantly because of diagnosis at an advanced stage of disease and a lack of effective treatments. We used the Gene Logic Inc. BioExpress platform and Affymetrix GeneChip arrays to identify genes differentially expressed in pancreatic cancer. cDNA was prepared from samples of normal pancreas (n = 11), normal gastrointestinal mucosa (n = 22), resected pancreas cancer tissues (n = 14), and pancreas cancer cell lines (n = 8), and was hybridized to the complete Affymetrix Human Genome U95 GeneChip set (arrays U95 A, B, C, D, and E) for simultaneous analysis of 60,000 cDNA fragments, with 12,000 fragments covering full-length genes and 48,000 fragments covering expressed sequence tags (ESTs). Genes expressed at levels at least fivefold greater in the pancreatic cancers ascompared to normal tissues were identified. Serial analysis of gene expression (SAGE) libraries (http://www.ncbi.nlm.nih.gov/SAGE/) of two normal pancreatic ductal cell cultures (HX and H126) were used to exclude genes expressed in the normal ducts (more than five tags per library). Differential expression of selected candidate genes was validated by immunohistochemical analysis (n = 3), by in situ hybridization (n = 1), and by reverse transcriptase-polymerase chain reaction (n = 8). One hundred eighty fragments were identified as having fivefold or greater expression levels in pancreas cancer specimens as compared to normal tissue, of which 124 corresponded to known genes and 56 to ESTs. Of these 124 fragments, 10 genes were represented by two or more fragments, resulting in 107 known genes identified as differentially expressed in pancreatic cancer. An additional 10 genes were expressed in the SAGE libraries of normal pancreatic duct epithelium, and were excluded from further analysis. A literature search indicated that 28 of the remaining 97 genes have been reported in association with pancreatic cancer, validating this approach. The remaining 69 genes have not been implicated in pancreatic cancer before, and have immediate potential as novel therapeutic targets and tumor markers of pancreatic cancer.

Chronic multifocal osteomyelitis, a new recessive mutation on chromosome 18 of the mouse.

Mice with tail kinks and deformities in their lower extremities were observed in a litter of C.D2-Qa-2+N6F15 mice. A mutant line that exhibits this phenotype in 100% of its offspring was established by subsequent breeding. The abnormalities resembled to some degree those found in a human syndrome termed chronic recurrent multifocal osteomyelitis (CRMO). Accordingly, we name the new mutation chronic multifocal osteomyelitis (cmo). Breeding analysis showed that the defect was determined by a single autosomal recessive gene. Restriction fragment length polymorphism (RFLP) analysis of progeny from a backcross between Mus musculus domesticus (CLA) and C.D2-Qa-(2+)-cmo/cmo indicated that the cmo gene resides on mouse Chromosome 18.

Disruption and Activation of the c-myb Locus by M-MuLV Insertion in Plasmacytoid Lymphosarcomas Induced by Pristane and Abelson Viruses

The c-myb proto-oncogene is the cellular homolog of the avian myeloblastosis virus oncogene (v-myb) (Roussel et al., 1979; Souza et al., 1980a; Bergman et al., 1981). The boundaries of c-myb are not yet known; however, studies have shown that the v-myb sequence is transduced from a portion of the coding region of c-myb into the avian myeloblastosis virus (AMV) (Klempnauer and Bishop 1983). AMV is a retrovirus that causes myeloblastic or monocytic leukemia in chickens and transforms myelomonocytic hematopoietic cells in culture (Moscovici 1975). The v-myb sequence is thought to be essential for the oncogenic potential of AMV (Duesberg et al., 1980; Souza 1980a; Gonda et al., 1981) and it appears that only certain target cells are responsive to the v-myb gene product (Moscovici 1975).

Alternate Forms of MYB: Consequences of Virus Insertion in Myeloid Tumorigenesis and Alternative Splicing in Normal Development

Nearly half of the proto-oncogenes were first identified in the genome of highly oncogenic avian and mammalian retroviruses (for review, see Bishop 1985). Several of these oncogenes appear to selectively transform cells of a particular lineage of differentiation. Studies on the v-myb containing avian myeloblastosis virus (AMV) and E-26 leukemia virus suggest that the viral oncogene v-myb is closely associated with myeloid tumorigenesis (for review, see Graf 1988). The transduction that gave rise to v-myb truncated the protooncogene c-myb at both of its ends. It is conceivable that myb may have multiple functions carried out by different structural domains. Removal of any of these domains may abolish either some function(s) performed by myb or its ability to be regulated by interaction with other cellular proteins. In order to understand the role of myb in normal and tumor development of myeloid cells, it is important to identify the various forms of myb in different cell types.

Structural Organization of Mouse c-myb Locus and the Mechanism of its Rearrangement in ABPL-2 Tumor Line Induced by Pristane and Abelson Murine Leukemia Virus

Abelson murine leukemia virus (A-MuLV) is a replication-defective transforming retrovirus that arose by recombination of nondefective helper virus (M-MuLV) and cellular sequences present within the normal mouse genome. The latter sequences, termed abl, appear to code for the transforming properties of the virus (for review see Rosenberg and Baltimore, 1980). This virus induces in adult BALB/c mice a variety of lymphoid neoplasms predominantly of the pre-B cell series (ABLS tumors). However, when the mice are previously injected with pristane, which induces intraperitonal granulomatous tissue, this virus also rapidly induces plasmacytomas (ABPC tumors) and occasionally, a morphological subset of lymphosarcomas characterized by plasmacytoid cytoplasm but with very little immunoglobulin production (ABPL tumors) (Potter et al.,1978). Our preliminary experiments indicated that ABPC and ABLS tumors produced abundant amounts of infectious A-MuLV particles while most ABPL tumors, in striking contrast, did not. In an effort to understand the molecular mechanisms involved in the genesis of these tumors, a detailed study of the expression of abl, myc and myb oncogenes was undertaken (Mushinski et al., 1983).