Thomas P. Dooley

Regulation of gene expression in inflammatory bowel disease and correlation with IBD drugs. Screening by DNA microarrays

Potential biomarkers for Crohns disease (CD) and ulcerative colitis (UC) were identified from two sets of full thickness pathologic samples utilizing DermArray® and PharmArray® DNA microarrays relative to uninvolved (Un) colon or normal colon. Seven of the over-expressed genes were verified using quantitative RT-PCR (i.e., TMPT, FABP1, IFI27, LCN2, COL11A2, HXB, and metallothionein). By correlating gene expression profiles between inflammatory bowel disease (IBD) tissue samples and IBD drug-treated cell cultures it might be possible to identify new candidate molecular target genes for IBD therapy and drug discovery. Potential biomarkers for CaCo2 cell cultures, which are routinely used as a GI tract surrogate model for in vitro pharmacokinetic studies, treated with azathioprine, 5-aminosalicylic acid, metronidazole, and prednisone were also identified from another experiment. Metallothionein mRNA expression was found to be down-regulated in azathioprine-treated CaCo2 cells, and was coincidentally up-regulated in the CD sample, thus resulting in an anti-correlation. These results suggest that this new screening methodology is feasible, that metallothioneins might be biomarkers for azathioprine therapy in vivo in CD, and that azathioprine might mechanistically down-regulate metallothionein gene expression. Correlations were also observed between IBD samples and either metronidazole- or 5-aminosalicylic acid-treated CaCo2 cells. Similar comparisons of disease tissue samples in vivo vs drug-treated cell cultures in vitro might reveal new mechanistic insights concerning established or experimental drug therapies. This affordable in vitro methodology is promising for expanded studies of IBD and other diseases.

Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR.

Squamous cell carcinoma (SCC) is the second most common form of skin cancer in Caucasians. Here we report on the identification of biomarkers of human cutaneous SCC cell lines in vitro and tissue samples in vivo using DermArray and PharmArray DNA microarrays, consisting of ca. 7400 unique human cDNAs. Differentially expressed genes were identified in two facial skin SCC cell lines (SCC 12 and SCC 13) compared to normal keratinocytes, and three cutaneous SCC tissue samples compared to normal skin. Quantitative validations of up- and down-regulated biomarkers were performed by qRT-PCR on 23 biomarker genes for the cell lines and 20 biomarker genes for the tumor tissues. In addition, three oral SCC cell lines were also included in the qRT-PCR validations for comparison, and the biomarker profiles were highly similar between the cutaneous and the oral SCC cell lines for all 23 biomarkers examined. The expression profiles for a variety of non-cutaneous SCC types, such as head-and-neck, oral, and lung, have been previously published. This report is the first to describe biomarkers for cutaneous SCC in two contexts, in vitro and in vivo. Although there was minimal overlap between the two different contexts using DNA microarrays, five genes were found common to both the cell lines and tissues, namely fibronectin 1, annexin A5, glyceraldehyde 3-phosphate dehydrogenase, zinc-finger protein 254, and huntingtin-associated protein interacting protein. Some of our previously published biomarkers of normal keratinocytes were down-regulated in SCC, suggestive of the dedifferentiated status of the transformed cells. While recent reports have identified some of the same genes as SCC biomarkers, for instance in head-and-neck cancer, thereby validating our approach, we have identified some novel biomarkers for cutaneous disease. These biomarker lists may be useful in molecular diagnostics of non-melanoma skin cancer, and a subset of the biomarkers might serve as suitable targets for drug discovery efforts of therapies for SCC.

Cell lines derived from ultraviolet radiation-induced benign melanocytic nevi in Monodelphis domestica exhibit cytogenetic aneuploidy

The gray short-tailed opossum, Monodelphis domestica, develops dermal melanocytic nevi (MN) after long-term chronic exposure to UVB (midwavelength ultraviolet radiation) alone. We developed cell lines from six UVB-induced dermal benign melanocytic lesion biopsies. One of the MN was determined histologically to be a benign melanoma (BM), whereas the remainder were benign melanocytic hyperplasias (MH). The cell lines were not tumorigenic when injected subcutaneously into athymic nude mice. Protein extracts prepared from these cell lines were analyzed electrophoretically on polyacrylamide gels and protease zymograms in preliminary attempts to identify protein and protease markers for pathogenesis. Cytogenetic analyses showed that half (three of six) of the MN cell lines exhibited aneuploidy involving extra copies of chromosomes 3, 5, 7, and/or 8. This result suggests that nonrandom aneuploidy can be an early event in chronic UVB induction of benign dermal melanocytic lesions. Karyotyping also showed a centromeric variant of chromosome 7 in some animals, which was confirmed to be constitutional. These Monodelphis cell lines will be valuable reagents for future studies of UVB-induced damage to mammalian skin.

cDNA Sequence of human p11 calpactin I light chain

The cDNA encoding full-length human p11 calpactin I light chain has been cloned and subjected to DNA sequencing. The open reading frame specifies a 97-amino-acid residue protein that surprisingly is identical to the p11 sequences of two mammalian ungulate species, cow and pig. However, the previously reported p11 polypeptide sequences of mouse and rat exhibited 8-9% nonidentity to human p11. These mammalian sequence comparison results are unexpected in view of current molecular cladistic theories that suggest a closer relationship between primates and rodents, rather than primates and ungulates. The mouse p11 gene has been previously mapped to chromosome 3 at a position syntenic with a centromeric-proximal region on human chromosome 1, and the human p11 cDNA clone is likely to be useful in physical mapping on chromosome 1.

UV-induced melanoma. A karyotype with a single translocation is stable after allografting and metastasis.

Metastatic melanoma cell lines were derived from a lymph node of a laboratory opossum, Monodelphis domestica, which had been exposed to mid-wavelength ultraviolet radiation (UVB) initially as a suckling young, and subsequently as a shaved juvenile and adult. The melanoma cell lines were dendritic and pigmented in vitro and contained a t(6;8)(p13;q13) as the only cytogenetic abnormality. The translocation was detected in 15% of primary cultures (passage 2) from the affected lymph node and in 100% of two ring-clone-derived lines, L1 and L2. The breakpoint or resulting partial trisomy of chromosomes 8 may have played a functional role in the tumorigenesis or metastasis of the tumor. The t(6;8) served as a convenient cytogenetic marker for allogeneic grafting studies in Monodelphis. The L2 cells were allografted subcutaneously (s.c.) into genetically diverse suckling young at 3 weeks of age and resulted in the growth of invasive, pigmented, primary and metastatic lesions affecting lymph nodes, lung, and other tissues. Metastatic variant cell lines, M1 and M3, were derived from the affected lungs of two animals and both lines demonstrated the same t(6;8), without additional numerical or structural chromosomal abnormalities. The maintenance of karyotypic stability with a single translocation during in vivo tumor growth and dissemination in this new allografting model is quiet remarkable, as most human metastatic melanomas exhibit multiple structural and numerical cytogenetic abnormalities.

A method to improve selection of molecular targets by circumventing the ADME pharmacokinetic system utilizing PharmArray DNA microarrays.

DNA microarrays may be used to identify potential molecular targets for drug discovery. Yet, DNA microarray experiments provide massive amounts of data. To limit the choice of potential molecular targets, it may be desirable to eliminate genes coincidentally up-regulated in tissues implicated in absorption, distribution, metabolism, and excretion (ADME) pharmacokinetics. DNA microarray experiments were performed to demonstrate a gene-exclusion approach using as an example RNA samples of neural origin, i.e., a human neuroblastoma cell line (SK-N-SH) and brain tissue, as the intended hypothetical site(s) of drug action. Biomarkers were identified using PharmArray DNA microarrays. The lists of neuroblastoma and neural biomarkers were constrained by limiting selection to the subset of genes that were not highly expressed in three transformed cell lines from liver, colon, and kidney (HepG2, Caco-2, and 786-O, respectively) that are routinely used as representatives of the ADME system during in vitro pharmacology and toxicology experiments. Principal component analysis methods with likelihood ratio-related bioinformatic tools were utilized to identify robust potential biomarker genes for the three ADME-related cell lines, neuroblastoma, and normal brain. Biomarkers of each sample were identified and selected genes were validated by qRT-PCR. Hundreds of biomarkers of the three ADME-related cell types, representing hepatocytes, kidney epithelium, and gastrointestinal tract, may now be used as a valuable database to restrict selection of biomarkers as potential molecular targets from the intended samples (e.g., neuroblastoma in this work). In addition to biomarker discovery per se, this demonstration suggests that our model method may be viable to help restrict gene lists during selection of potential molecular targets for subsequent drug discovery.

Methods for gene expression profiling in dermatology research using DermArray nylon filter DNA microarrays.

Here we present methods of gene expression profiling using nylon filter deoxyribonucleic acid (DNA) microarrays and radiolabeled and nonradiolabeled hybridization probes. DermArray(R) nylon filter DNA microarrays were designed specifically for use in dermatology research. A patent-pending method was used to select approx 4400 highly informative, sequence-verified human cDNA clones for this DNA micro array. Using DermArray(R) filters, biomarkers have been discovered for normal and pathologic cells from skin, and for responses to dermatologic drugs. As an example, gene expression profiling was performed with hydroquinone-treated SKMel-28 cells, a melanoma cell line. Also included are the methods for bioinformatic analysis using Pathwaystrade mark software.