Differential Gene Expression Analysis by DNA Microarray Technology and Its Application in Molecular Oncology

Accumulation of genetic and epigenetic aberrations leads to malignant transformation of normal cells. Functional studies of cancer using genomic and proteomic tools aim to reveal the true complexity of the processes leading to cancer development in humans. Until recently, diagnosis and prognosis of cancer was based on conventional pathologic criteria and epidemiological evidence. Certain tumors were divided only into relatively broad histological and morphological subcategories. Rapidly developing methods of differential gene expression analysis promote the search for clinically relevant genes changing their expression levels during malignant transformation. DNA microarrays offer a unique possibility to rapidly assess the global expression picture of thousands genes in any given time point and compare the results of detailed combinatory analysis of global expression profiles for normal and malignant cells at various functional stages or separate experimental conditions. Acquisition of such genetic portraits allows searching for regularity and difference in expression patterns of certain genes, understanding their function and pathological importance, and ultimately developing the molecular nosology of cancer. This review describes the basis of DNA microarray technology and methodology, and focuses on their application in molecular classification of tumors, drug sensitivity and resistance studies, and identification of biological markers of cancer.     

Immunochemical and molecular-Genetic markers in diagnostics of gastric cancer

Intensive studies of molecular mechanisms responsible for tumor transformation results in identification of new proteins and their genes involved into tumor development. These proteins may be used as markers of tumor transformation of cells and the level of their expression may be evaluated by means of modern highly sensitive and technological methods of analysis. This review summarized literature data on currently used immunohistochemical and molecular genetic markers of gastric cancer. It highlights genetic and epigenetic changes detected in nucleic acids of tumor tissue cells in malignant and benign gastric diseases as well as in the level of DNA circulating in blood of patients with gastric cancer.     

Interaction between APC and Fen1 during breast carcinogenesis

Aberrant DNA base excision repair (BER) contributes to malignant transformation. However, inter-individual variations in DNA repair capacity plays a key role in modifying breast cancer risk. We review here emerging evidence that two proteins involved in BER – adenomatous polyposis coli (APC) and flap endonuclease 1 (Fen1) – promote the development of breast cancer through novel mechanisms. APC and Fen1 expression and interaction is increased in breast tumors versus normal cells, APC interacts with and blocks Fen1 activity in Pol-β-directed LP-BER, and abrogation of LP-BER is linked with cigarette smoke condensate-induced transformation of normal breast epithelial cells. Carcinogens increase expression of APC and Fen1 in spontaneously immortalized human breast epithelial cells, human colon cancer cells, and mouse embryonic fibroblasts. Since APC and Fen1 are tumor suppressors, an increase in their levels could protect against carcinogenesis; however, this does not seem to be the case. Elevated Fen1 levels in breast and lung cancer cells may reflect the enhanced proliferation of cancer cells or increased DNA damage in cancer cells compared to normal cells. Inactivation of the tumor suppressor functions of APC and Fen1 is due to their interaction, which may act as a susceptibility factor for breast cancer. The increased interaction of APC and Fen1 may occur due to polypmorphic and/or mutational variation in these genes. Screening of APC and Fen1 polymorphic and/or mutational variations and APC/Fen1 interaction may permit assessment of individual DNA repair capability and the risk for breast cancer development. Such individuals might lower their breast cancer risk by reducing exposure to carcinogens. Stratifying individuals according to susceptibility would greatly assist epidemiologic studies of the impact of suspected environmental carcinogens. Additionally, a mechanistic understanding of the interaction of APC and Fen1 may provide the basis for developing new and effective targeted chemopreventive and chemotherapeutic agents.     

DNA Demethylation and Carcinogenesis

DNA methylation plays an important role in the establishment and maintenance of the program of gene expression. Tumor cells are characterized by a paradoxical alteration of DNA methylation pattern: global DNA demethylation and local hypermethylation of certain genes. Hypermethylation and inactivation of tumor suppressor genes are well documented in tumors. The role of global genome demethylation in carcinogenesis is less studied. New data provide evidence for independence of DNA hypo- and hypermethylation processes in tumor cells. These processes alter expression of genes that have different functions in malignant transformation. Recent studies have demonstrated that global decrease in the level of DNA methylation is related to hypomethylation of repeated sequences, increase in genetic instability, hypomethylation and activation of certain genes that favor tumor growth, and increase in their metastatic and invasive potential. The recent data on the role of DNA demethylation in carcinogenesis are discussed in this review. The understanding of relationships between hypo- and hypermethylation in tumor cells is extremely important due to reversibility of DNA methylation and attempts to utilize for anti-tumor therapy the drugs that modify DNA methylation pattern.__________Translated from Biokhimiya, Vol. 70, No. 7, 2005, pp. 900–911.Original Russian Text Copyright © 2005 by Kisseljova, Kisseljov.This article was not published in the journal special issue devoted to the 70th anniversary of B. F. Vanyushin (Biochemistry (Moscow) (2005) 70, No. 5) because of the limiting volume of the journal.     

Bioinformatics analysis of the gene expression profile in Bladder carcinoma

Bladder carcinoma, which has the ninth highest incidence among malignant tumors in the world, is a complex, multifactorial disease. The malignant transformation of bladder cells results from DNA mutations and alterations in gene expression levels. In this work, we used a bioinformatics approach to investigate the molecular mechanisms of bladder carcinoma. Biochips downloaded from the Gene Expression Omnibus (GEO) were used to analyze the gene expression profile in urinary bladder cells from individuals with carcinoma. The gene expression profile of normal genomes was used as a control. The analysis of gene expression revealed important alterations in genes involved in biological processes and metabolic pathways. We also identified some small molecules capable of reversing the altered gene expression in bladder carcinoma; these molecules could provide a basis for future therapies for the treatment of this disease.     

Bracken fern-induced malignant tumors in rats: absence of mutations in p53, H-ras and K-ras and no microsatellite instability

Bracken fern (genus Pteridium) has been shown to induce tumors in domestic and experimental animals. Epidemiological studies have also shown an association between human exposure to bracken toxins and increased risk for the development of upper gastrointestinal tract tumors. Our aim in this study was to investigate possible genomic alterations in bracken fern-induced tumors of experimental animals searching for molecular markers that might be used for human epidemiological studies. Using human colorectal carcinogenesis as a molecular model, we examined eight malignant bracken fern-induced tumors of rats for mutations in the genes associated with the "classic pathway" of colorectal cancer, i.e. p53 and ras, and also in the "mutator pathway" by evaluating microsatellite instability. Exons 5-9 of the p53 gene and exons 1 and 2 of the K-ras and H-ras genes were examined by DNA sequencing and no mutations were found in any of the eight tumors. Amplification of five previously validated microsatellite loci (one with mono-, three with di- and one with tetra-nucleotide repeat motifs) in the malignant tumors and in the surrounding normal tissue did not reveal any instability. The involvement of epigenetic alterations or of mutations in other tumor suppressor genes or oncogenes should be further investigated in the search for human epidemiological markers.     

Overlapping Genes May Control Reprogramming of Mouse Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) and Breast Cancer Stem Cells

Recent findings suggest the possibility that tumors originate from cancer cells with stem cell properties. The cancer stem cell (CSC) hypothesis provides an explanation for why existing cancer therapies often fail in eradicating highly malignant tumors and end with tumor recurrence. Although normal stem cells and CSCs both share the capacity for self-renewal and multi-lineage differentiation, suggesting that CSC may be derived from normal SCs, the cellular origin of transformation of CSCs is debatable. Research suggests that the tightly controlled balance of self-renewal and differentiation that characterizes normal stem cell function is dis-regulated in cancer. Additionally, recent evidence has linked an embryonic stem cell (ESC)-like gene signature with poorly differentiated high-grade tumors, suggesting that regulatory pathways controlling pluripotency may in part contribute to the somatic CSC phenotype. Here, we introduce expression profile bioinformatic analyses of mouse breast cells with CSC properties, mouse embryonic stem (mES) and induced pluripotent stem (iPS) cells with an emphasis on how study of pluripotent stem cells may contribute to the identification of genes and pathways that facilitate events associated with oncogenesis. Global gene expression analysis from CSCs and induced pluripotent stem cell lines represent an ideal model to study cancer initiation and progression and provide insight into the origin cancer stem cells. Additionally, insight into the genetic and epigenomic mechanisms regulating the balance between self-renewal and differentiation of somatic stem cells and cancer may help to determine whether different strategies used to generate iPSCs are potentially safe for therapeutic use.     

Cell cycle alterations and lung cancer

It is now widely accepted that human carcinogenesis is a multi-step process and phenotypic changes during cancer progression reflect the sequential accumulation of genetic alterations in cells. The recent progress of scientific research has notably increased knowledge about biological events involved in lung cancer pathogenesis and progression, thanks to the use of molecular biology and immunohistochemistry techniques. Lots of the genetic alteration found in small cells lung cancer (SCLC) and in not small cells lung cancer (NSCLC) concern the expression of cell cycle genes, actually recognized as onco-suppressor genes and the lack of equilibrium between oncogenes and oncosuppressor genes. The present review of literature widely describes the cell cycle control, the lung cancer molecular pathogenesis, the catalog of known genetic alterations and the recent advances in global expression profiles in lung tumors, on the basis of the various hystological types too. Such data suggest the potential use of this knowledges in clinical practice both as prognostic factors and innovative therapeutic possibilities and they impose the necessity of new studies about cell cycle control and lung carcinogenesis.     

Immortalized cells as experimental models to study cancer

The development of cancer is a multi-step process in which normal cells sustain a series of genetic alterations that together program the malignant phenotype. Much of our knowledge of cancer biology results from the detailed study of specimens and cell lines derived from patient tumors. While these approaches continue to yield critical information regarding the identity, number, and types of alterations found in human tumors, further progress in understanding the molecular basis of malignant transformation depends upon the generation and use of increasingly sophisticated experimental models of cancer. Over the past several years, the recognition that telomeres and telomerase play essential roles in regulating cell lifespan now permits the development of new models of human cancer. Here we review recent progress in the use of immortalized human cells as a foundation for understanding the molecular basis of cancer.     

Comprehensive genetic analysis of cancer cells

Human cancer is viewed as a disorder of genes originating from the progeny of a single cell that has accumulated multiple genetic alterations. The genetic alterations include point mutation, chromosomal rearrangements and imbalances. Amplifications primarily involve oncogenes whose overexpression leads to growth deregulation, while deletions commonly target tumor suppressor genes that control cell cycle checkpoints and DNA repair mechanisms. With the advent of molecular cytogenetics procedures for global detection of genomic imbalances and for multicolor visualization of structural chromosome changes, as well as the completion of human genome mapping and the development of microarray technology for serial gene expression analysis of the entire genomes, a significant progress has been made in uncovering the molecular basis of cancer. The major challenge in cancer biology is to decipher the molecular anatomy of various cancers and to identify cancer-related genes that now comprise only a fraction of human genes. The complete genetic anatomy of specific cancers would allow a better understanding of the role of genetic alterations in carcinogenesis, provide diagnostic and prognostic markers and discriminate between cells at different stages of progression toward malignancy. This review highlights current technologies that are available to explore cancer cells and outlines their application to investigations in human hepatocellular carcinoma.     

A DNA microarray survey of gene expression in normal human tissues

Background  

Numerous studies have used DNA microarrays to survey gene expression in cancer and other disease states. Comparatively little is known about the genes expressed across the gamut of normal human tissues. Systematic studies of global gene-expression patterns, by linking variation in the expression of specific genes to phenotypic variation in the cells or tissues in which they are expressed, provide clues to the molecular organization of diverse cells and to the potential roles of the genes.     

Recent advances in cancer research: mouse models of tumorigenesis

Over the past 20 years, cancer research has gained major insights into the complexity of tumor development, in particular into the molecular mechanisms that underlie the progressive transformation of normal cells into highly malignant derivatives. It is estimated that the transformation of a normal cell to a malignant tumor cell is dependent upon a small number of genetic alterations, estimated to be within the range of four to seven rate-limiting events. Critical events in the evolution of neoplastic disease include the loss of proliferative control, the failure to undergo programmed cell death (apoptosis), the onset of neoangiogenesis, tissue remodeling, invasion of tumor cells into surrounding tissue and, finally, metastatic dissemination of tumor cells to distant organs. In patients, the molecular analysis of these multiple steps is hampered by the unavailability of tumor biopsies from all tumor stages. In contrast, mouse models of tumorigenesis allow the reproducible isolation of all tumor stages, including normal tissue, which are then amenable to pathological, genetic and biochemical analyses and, hence, have been instrumental in investigating cancer-related genes and their role in carcinogenesis. In this review, we discuss mouse tumor models that have contributed substantially to the identification and characterization of novel tumor pathways. In particular, we focus on transgenic and knockout mouse models that closely mimic human cancer and thus can be used as model systems for cancer research.