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Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

An unsuspected finding in cancer research is that different cancers result from mutations in different genes. If cancer is a problem of multicellularity, why is there not a single set of genes regulating all cancers of multicellular animals? Cancer risk is expected to be higher in larger, longer-lived species since the risk of cancer-initiating somatic mutations is expected to increase with the number of lifetime cell divisions. However, this expected relationship is not observed, and has been termed Peto’s Paradox. The evolution of additional mechanisms of cancer suppression in large, long-lived species can resolve the paradox and may explain the differences in the genetics of cancer between species and tissues. In a proof-of-concept experimental evolution study fruit flies with hereditary tumors were selected to evolve suppression of these tumors. A significant reduction in the incidence of tumors was observed in the final populations. The genetic changes involved in the evolution of additional mechanisms may come in two forms. First, a cancer-suppression mechanism acting may be recruited by its upregulation of gene expression in the target tissue. To address this, I compared the levels of gene expression of 15 tumor suppressor genes and 8 proto-oncogenes across dozens of non- diseased human tissues. I found that 15 of the 23 genes have their highest level of expression in the tissue types where they are implicated in hereditary cancer, relative to the tissue types where they are not. Second, additional mechanisms may arise by duplications of pre-existing tumor suppressor genes already acting in the target tissue. The recent finding of additional copies of the critical tumor suppressor gene, TP53, in the large-bodied African Elephant and the long-lived microbats has been suggested to explain the low-rates of cancers in these species. However, in a phylogenetic comparison of the coding regions of these retrogene copies relative to the normal TP53 copies in 24 mamma

An unsuspected finding in cancer research is that different cancers result from mutations in different genes. If cancer is a problem of multicellularity, why is there not a single set of genes regulating all cancers of multicellular animals? Cancer risk is expected to be higher in larger, longer-lived species since the risk of cancer-initiating somatic mutations is expected to increase with the number of lifetime cell divisions. However, this expected relationship is not observed, and has been termed Peto’s Paradox. The evolution of additional mechanisms of cancer suppression in large, long-lived species can resolve the paradox and may explain the differences in the genetics of cancer between species and tissues. In a proof-of-concept experimental evolution study fruit flies with hereditary tumors were selected to evolve suppression of these tumors. A significant reduction in the incidence of tumors was observed in the final populations. The genetic changes involved in the evolution of additional mechanisms may come in two forms. First, a cancer-suppression mechanism acting may be recruited by its upregulation of gene expression in the target tissue. To address this, I compared the levels of gene expression of 15 tumor suppressor genes and 8 proto-oncogenes across dozens of non- diseased human tissues. I found that 15 of the 23 genes have their highest level of expression in the tissue types where they are implicated in hereditary cancer, relative to the tissue types where they are not. Second, additional mechanisms may arise by duplications of pre-existing tumor suppressor genes already acting in the target tissue. The recent finding of additional copies of the critical tumor suppressor gene, TP53, in the large-bodied African Elephant and the long-lived microbats has been suggested to explain the low-rates of cancers in these species. However, in a phylogenetic comparison of the coding regions of these retrogene copies relative to the normal TP53 copies in 24 mamma

Cytogenetic aberrations, such as chromosomal translocations, aneuploidy, and amplifications, are frequently detected in hematological malignancies. For many of the common autosomal aberrations, the mechanisms underlying their roles in cancer development have been well-characterized. On the contrary, although loss of a sex chromosome is observed in a broad range of hematological malignancies, how it cooperates in disease development is less understood. Nevertheless, it has been postulated that tumor suppressor genes reside on the sex chromosomes. Although the X and Y sex chromosomes are highly divergent, the pseudoautosomal regions are homologous between both chromosomes. Here, we review what is currently known about the pseudoautosomal region genes in the hematological system. Additionally, we discuss implications for haploinsufficiency of critical pseudoautosomal region sex chromosome genes, driven by sex chromosome loss, in promoting hematological malignancies. Because mechanistic studies on disease development rely heavily on murine models, we also discuss the challenges and caveats of existing models, and propose alternatives for examining the involvement of pseudoautosomal region genes and loss of a sex chromosome in vivo. With the widespread detection of loss of a sex chromosome in different hematological malignances, the elucidation of the role of pseudoautosomal region genes in the development and progression of these diseases would be invaluable to the field.

BackgroundStudies of familial cancers have found that only a small subset of tissues are affected by inherited mutations in a given tumour suppressor gene (TSG) or proto-oncogene (POG), even though the mutation is present in all tissues. Previous tests have shown that tissue specificity is not due to the presence vs absence of gene expression, as TSGs and POGs are expressed in nearly every type of normal human tissue. Using published microarray expression data we tested the related hypothesis that tissue-specific expression of a TSG or POG is highest in tissue where it is of oncogenic importance.MethodsWe tested this hypothesis by examining whether individual TSGs and POGs had higher expression in the normal (noncancerous) tissues where they are implicated in familial cancers relative to those tissues where they are not. We examined data for 15 TSGs and 8 POGs implicated in familial cancer across 12 human tissue types.ResultsWe found a significant difference between expression levels in susceptible vs nonsusceptible tissues. It was found that 9 (60%, P<0.001) of the TSGs and 5 (63%, P<0.001) of the POGs had their highest expression level in the tissue type susceptible to their oncogenic effect.ConclusionsThis highly significant association supports the hypothesis that mutation of a specific TSG or POG is likely to be most oncogenic in the tissue where the gene has its highest level of expression. This suggests that high expression in normal tissues is a potential marker for linking cancer-related genes with their susceptible tissues.

BackgroundStudies of familial cancers have found that only a small subset of tissues are affected by inherited mutations in a given tumour suppressor gene (TSG) or proto-oncogene (POG), even though the mutation is present in all tissues. Previous tests have shown that tissue specificity is not due to the presence vs absence of gene expression, as TSGs and POGs are expressed in nearly every type of normal human tissue. Using published microarray expression data we tested the related hypothesis that tissue-specific expression of a TSG or POG is highest in tissue where it is of oncogenic importance.MethodsWe tested this hypothesis by examining whether individual TSGs and POGs had higher expression in the normal (noncancerous) tissues where they are implicated in familial cancers relative to those tissues where they are not. We examined data for 15 TSGs and 8 POGs implicated in familial cancer across 12 human tissue types.ResultsWe found a significant difference between expression levels in susceptible vs nonsusceptible tissues. It was found that 9 (60%, P<0.001) of the TSGs and 5 (63%, P<0.001) of the POGs had their highest expression level in the tissue type susceptible to their oncogenic effect.ConclusionsThis highly significant association supports the hypothesis that mutation of a specific TSG or POG is likely to be most oncogenic in the tissue where the gene has its highest level of expression. This suggests that high expression in normal tissues is a potential marker for linking cancer-related genes with their susceptible tissues.

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

The purpose of this research is to demonstrate that gene amplification of HER-2/neu is associated with loss of expression of the tetraspanin metastasis suppressor gene, KAI1/CD82, in breast cancer cell lines and patients' primary breast tumors. The scope is limited to breast cancer and mechanisms of metastasis. Results: 1) We identified three cell lines with gene amplification of HER-2/neu and negative KAI1/CD82 expression to serve as model systems and corroborated these results in primary breast tumors. 2) We found that CO-029, a tetraspanin metastasis promoter gene, was up-regulated in HER-2/neu+ lines. 3) We ectopically expressed HER-2/neu in MCF-7 cells and demonstrated that over-expression of HER-2/neu resulted in loss of expression of KAI1/CD82, but increased expression of CO-029. 4) Finally, we knocked down expression of HER-2/neu by RNAi and demonstrated that KAI1/CD82 levels increased four-fold. Significance: HER-2/neu signaling regulates tetraspanin gene expression to promote metastatic potential. This novel mechanism may explain why women with gene amplification of HER-2/neu in their breast tumors are at higher risk for metastasis and death. This is the first demonstration of a receptor tyrosine kinase (RTX) that regulates tetraspanin metastasis genes, and this may be the first example of a broader paradigm linking RTK oncogenes to metastasis genes.

MLL amplification was recently recognized as a recurrent aberration in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), associated with adverse prognosis and karyotype complexity. Here we present detailed results of fluorescence in situ hybridization (FISH) and expression analyses of MLL and 5 selected 11q candidate oncogenes (CBL, DDX6, ETS1, FLI1, and PLZF) in 31 patient samples and one cell line with 11q23 gain. FISH analyses revealed that the 11q23 amplicon invariably encompassed MLL, DDX6, ETS1, and FLI1, whereas expression analyses identified MLL and DDX6 as the most differentially expressed genes among samples with and without 11q23 copy gain or amplification. In MLL-amplified samples, a significant transcriptional up-regulation of MEIS1, PROML1, ADAM10, NKG2D, and ITPA was noted. Further analyses, designed to elucidate a possible role of the 11q overexpressed genes (MLL, DDX6, FLI1, and ETS1) in unselected MDS and AML samples, revealed a significant upregulation of MLL in MDS. Our findings confirm the MLL gene as a prominent target of 11q23 amplification and provide further evidence for an etiologic role for MLL gain of function in myeloid malignancies. In addition, our results indicate that the transcriptional program associated with MLL rearrangements and MLL overexpression displays significant similarities.

Rearrangements of the ALL-1/MLL1 gene underlie the majority of infant acute leukaemias, as well as of therapy-related leukaemias developing in cancer patients treated with inhibitors of topoisomerase II, such as VP16 and doxorubicin. The rearrangements fuse ALL-1 to any of >50 partner genes or to itself. Here, we describe the unique features of ALL-1-associated leukaemias, and recent progress in understanding molecular mechanisms involved in the activity of the ALL-1 protein and of its Drosophila homologue TRITHORAX.

Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of mixed lineage leukemia (MLL) gene rearrangements, a poor outcome, and resistance to chemotherapeutic drugs. One exception is cytosine arabinoside (Ara-C), to which infant ALL cells are highly sensitive. To investigate the mechanism underlying Ara-C sensitivity in infants with ALL, mRNA levels of Ara-C-metabolizing enzymes were measured in infants (n = 18) and older children (noninfants) with ALL (n = 24). In the present study, infant ALL cells were 3.3-fold more sensitive to Ara-C (P =.007) and accumulated 2.3-fold more Ara-CTP (P =.011) upon exposure to Ara-C, compared with older children with ALL. Real-time quantitative reverse trancriptase-polymerase chain reaction (RT-PCR) (TaqMan) revealed that infants express 2-fold less of the Ara-C phosphorylating enzyme deoxycytidine kinase (dCK) mRNA (P =.026) but 2.5-fold more mRNA of the equilibrative nucleoside transporter 1 (hENT1), responsible for Ara-C membrane transport (P =.001). The mRNA expression of pyrimidine nucleotidase I (PN-I), cytidine deaminase (CDA), and deoxycytidylate deaminase (dCMPD) did not differ significantly between both groups. hENT1 mRNA expression inversely correlated with in vitro resistance to Ara-C (r(s) = -0.58, P =.006). The same differences concerning dCK and hENT1 mRNA expression were observed

The proto-oncogene EVI1 encodes a DNA binding protein and is located on chromosome 3q26. The gene is aberrantly expressed in acute myeloid leukemia (AML) patients carrying 3q26 abnormalities. Two mRNAs are transcribed from this locus: EVI1 and a fusion of EVI1 with MDS1 (MDS1-EVI1), a gene located 5' of EVI1. The purpose of this study was to investigate which of the 2 gene products is involved in transformation in human AML. To discriminate between EVI1 and MDS1-EVI1 transcripts, distinct real-time quantitative polymerase chain reaction (PCR) assays were developed. Patients with 3q26 abnormalities often showed high EVI1 and MDS1-EVI1 expression. In a cohort of 319 AML patients, 4 subgroups could be distinguished: EVI1(+) and MDS1-EVI1(-) (6 patients; group I), EVI1(+) and MDS1-EVI1(+) (26 patients; group II), EVI1(-) and MDS1-EVI1(+) (12 patients; group III), and EVI1(-) and MDS1-EVI1(-) (275 patients; group IV). The only 4 patients with a 3q26 aberration belonged to groups I and II. Interestingly, high EVI1 and not MDS1-EVI1 expression was associated with unfavorable karyotypes (eg, -7/7q-) or complex karyotypes. Moreover, a significant correlation was observed between EVI1 expression and 11q23 aberrations (mixed lineage leukemia [MLL] gene involvement). Patients from groups I and II had significantly shorter overall and event-free survival than patients in groups III and IV. Our data demonstrate that high EVI1 expression is an independent poor prognostic marker within the intermediate- risk karyotypic group.

Li Ching-wan., Thesis submitted in: November 2001., Thesis (M.Phil.)--Chinese University of Hong Kong, 2002., Includes bibliographical references (leaves 104-130)., s in English and Chinese., ACKNOWLEDGEMENTS --- p.i, S (ENGLISH/CHINESE) --- p.iii, LIST OF FIGURES --- p.xi, LIST OF TABLES --- p.xiii, LIST OF ABBREVIATIONS --- p.xiv, Chapter CHAPTER1 --- INTRODUCTION, Chapter 1.1. --- Liver Cancer --- p.1, Chapter 1.2. --- Hepatocellular Carcinoma --- p.1, Chapter 1.2.1. --- Types of Liver Cancer --- p.1, Chapter 1.2.2. --- Epidemiology --- p.4, Chapter 1.2.2.1. --- Geographical Distribution --- p.4, Chapter 1.2.2.2. --- Age and Gender Distribution --- p.8, Chapter 1.2.3. --- Etiologic Factors --- p.9, Chapter 1.2.3.1. --- Chronic Infection with Hepatitis B (HBV) and C (HCV) Viruses --- p.9, Chapter 1.2.3.2. --- Aflatoxin B1 --- p.11, Chapter 1.2.3.3. --- Alcohol --- p.12, Chapter 1.2.3.4. --- Summary --- p.12, Chapter 1.3. --- HCC in Hong Kong --- p.14, Chapter 1.4. --- Role of Viral Hepatitis B in HCC --- p.17, Chapter 1.4.1. --- HBV Genome --- p.17, Chapter 1.4.2. --- Consequences of HBV DNA Integration --- p.17, Chapter 1.4.2.1. --- HBV Integration --- p.17, Chapter 1.4.2.2. --- Transactivation of Cellular Genes by HBV DNA --- p.19, Chapter 1.4.2.3. --- Chromosomal DNA Instability --- p.20, Chapter 1.5. --- Genetic Alterations in HCC --- p.21, Chapter 1.5.1. --- Tumor Suppressor Gene --- p.21, Chapter 1.5.2. --- Proto-oncogene --- p.23, Chapter 1.5.3. --- Genetic Studies in HCC --- p.23, Chapter 1.5.3.1. --- Loss of Heterozygosity (LOH) --- p.25, Chapter 1.5.3.2. --- Comparative Genomic Hybridization (CGH) --- p.26, Chapter 1.5.3.3. --- Array CGH --- p.26, Chapter 1.5.4. --- Large-Scale Genetic Analysis in HCC --- p.27, Chapter CHAPTER2 --- RATIONALE IN THIS STUDY --- p.35, Chapter CHAPTER3 --- MATERIALS AND METHODS, Chapter 3.1. --- Patients and Materials --- p.38, Chapter 3.1.1. --- DNA Extraction --- p.40, Chapter 3.2. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.40, Chapter 3.2.1. --- Microsatellite Markers --- p.41, Chapter 3.2.2. --- Amplification of Target Sequences by PCR --- p.42, Chapter 3.2.2.1. --- 5-end Labeling Primers --- p.42, Chapter 3.2.2.2. --- Amplification of Target Sequences --- p.42, Chapter 3.2.3. --- Denaturing Polyacrylamide Gel --- p.44, Chapter 3.2.3.1. --- Electrophoresis --- p.44, Chapter 3.2.4. --- Detection of Loss of Heterozygosity (LOH) --- p.45, Chapter 3.2.5. --- Duplex PCR Analysis of Homozygous Deletion --- p.45, Chapter 3.3. --- Amplification Analysis by Array-CGH --- p.46, Chapter 3.3.1. --- Nick-Translation --- p.49, Chapter 3.3.2. --- Hybridization --- p.49, Chapter 3.3.3. --- Imaging and Data Analysis --- p.50, Chapter 3.3.4. --- Determination of Normal Range for All Cases --- p.51, Chapter 3.3.5. --- Assessment of Data Quality --- p.51, Chapter 3.4. --- Statistical Analysis --- p.52, Chapter CHAPTER4 --- RESULTS, Chapter 4.1. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.53, Chapter 4.1.1. --- Region I of Smallest Common Deletion Region --- p.54, Chapter 4.1.2. --- Region II of Smallest Common Deletion Region --- p.54, Chapter 4.2. --- Amplification Analysis by Array-CGH --- p.62, Chapter CHAPTER5 --- DISCUSSION, Chapter 5.1. --- LOH Analysis on Chromosome 4q --- p.73, Chapter 5.1.1. --- LOH of Chromosome 4q in Various Cancers --- p.74, Chapter 5.1.1.1. --- Hepatocellular Carcinomas --- p.74, Chapter 5.1.1.2. --- Other Neoplasia --- p.76, Chapter 5.1.2. --- Functional Studies on Chromosome 4 --- p.76, Chapter 5.1.3. --- Putative Tumor Suppressors on Chromosome 4q --- p.80, Chapter 5.1.3.1. --- Region I (4q27-q28.1) --- p.80, Chapter 5.1.3.1.1. --- MAD2L1 (4q27) --- p.80, Chapter 5.1.3.2. --- Region II (4q35.2) --- p.81, Chapter 5.1.3.2.1. --- INGlL(4q35.1) --- p.81, Chapter 5.1.3.2.2. --- FAT (4q34-q35) --- p.81, Chapter 5.1.3.2.3. --- Caspase 3 (4q35) --- p.82, Chapter 5.1.4. --- Limitation of this Study --- p.83, Chapter 5.1.4.1. --- Markers --- p.83, Chapter 5.1.4.1.1. --- Limitation of the Markers --- p.83, Chapter 5.1.4.1.2. --- Location of the Microsatellite Markers --- p.83, Chapter 5.1.4.2. --- Tissue Samples --- p.84, Chapter 5.1.4.2.1. --- Normal Reference --- p.84, Chapter 5.1.4.2.2. --- Pathologic Characterization --- p.85, Chapter 5.1.5. --- Future Studies --- p.85, Chapter 5.1.5.1. --- Improvement of the Experiment --- p.85, Chapter 5.1.5.2. --- Extension of the Present Study --- p.86, Chapter 5.2. --- Amplification Analysis by Array-CGH --- p.88, Chapter 5.2.1. --- Amplicons Showing Amplification in HCC --- p.89, Chapter 5.2.1.1. --- Locus of 17q23 --- p.89, Chapter 5.2.1.1.1. --- D17S1670 --- p.89, Chapter 5.2.1.1.2. --- RPS6KB1 --- p.91, Chapter 5.2.1.2. --- Locus of 1q25-q31 --- p.92, Chapter 5.2.1.2.1. --- LAMC2 --- p.92, Chapter 5.2.1.3. --- Locus of 3q26.3 --- p.93, Chapter 5.2.1.3.1. --- PIK3CA --- p.93, Chapter 5.2.1.4. --- Locus of 8p22 --- p.94, Chapter 5.2.1.4.1. --- CTSB --- p.94, Chapter 5.2.1.5. --- Locus of 6q22 --- p.95, Chapter 5.2.1.5.1. --- MYB --- p.95, Chapter 5.2.1.6. --- Locus of 20ql3 --- p.96, Chapter 5.2.1.6.1. --- CSE1L --- p.96, Chapter 5.2.1.7. --- Locus of Ip36.2-p35.1 --- p.97, Chapter 5.2.1.7.1. --- FGR --- p.97, Chapter 5.2.1.8. --- Locus of 7q21.1 --- p.98, Chapter 5.2.1.8.1. --- PGY1 --- p.98, Chapter 5.2.2. --- Amplicons Showing Deletion in HCC --- p.99, Chapter 5.2.2.1. --- Loss at 11ql3 and 14q32.3 --- p.99, Chapter 5.2.3. --- Limitation of the Study --- p.100, Chapter 5.2.3.1. --- Samples and Materials --- p.100, Chapter 5.2.4. --- Further Study --- p.101, Chapter 5.2.4.1. --- Confirmation of the Result in Various Levels --- p.101, Chapter 5.2.4.2. --- Assessment of the Significant Losses on Chromosomes 11ql3 and 14ql3 --- p.101, Chapter 5.2.5. --- Application of Microarray in Genetic Studies --- p.102, Chapter 5.2.5.1. --- Deletion Analysis --- p.102, Chapter 5.2.5.2 --- Tissue Microarray --- p.103, Chapter 5.2.5.3. --- cDNA Microarray --- p.103, Chapter chapter6 --- references --- p.104, http://library.cuhk.edu.hk/record=b5891353, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Li Ching-wan., Thesis submitted in: November 2001., Thesis (M.Phil.)--Chinese University of Hong Kong, 2002., Includes bibliographical references (leaves 104-130)., s in English and Chinese., ACKNOWLEDGEMENTS --- p.i, S (ENGLISH/CHINESE) --- p.iii, LIST OF FIGURES --- p.xi, LIST OF TABLES --- p.xiii, LIST OF ABBREVIATIONS --- p.xiv, Chapter CHAPTER1 --- INTRODUCTION, Chapter 1.1. --- Liver Cancer --- p.1, Chapter 1.2. --- Hepatocellular Carcinoma --- p.1, Chapter 1.2.1. --- Types of Liver Cancer --- p.1, Chapter 1.2.2. --- Epidemiology --- p.4, Chapter 1.2.2.1. --- Geographical Distribution --- p.4, Chapter 1.2.2.2. --- Age and Gender Distribution --- p.8, Chapter 1.2.3. --- Etiologic Factors --- p.9, Chapter 1.2.3.1. --- Chronic Infection with Hepatitis B (HBV) and C (HCV) Viruses --- p.9, Chapter 1.2.3.2. --- Aflatoxin B1 --- p.11, Chapter 1.2.3.3. --- Alcohol --- p.12, Chapter 1.2.3.4. --- Summary --- p.12, Chapter 1.3. --- HCC in Hong Kong --- p.14, Chapter 1.4. --- Role of Viral Hepatitis B in HCC --- p.17, Chapter 1.4.1. --- HBV Genome --- p.17, Chapter 1.4.2. --- Consequences of HBV DNA Integration --- p.17, Chapter 1.4.2.1. --- HBV Integration --- p.17, Chapter 1.4.2.2. --- Transactivation of Cellular Genes by HBV DNA --- p.19, Chapter 1.4.2.3. --- Chromosomal DNA Instability --- p.20, Chapter 1.5. --- Genetic Alterations in HCC --- p.21, Chapter 1.5.1. --- Tumor Suppressor Gene --- p.21, Chapter 1.5.2. --- Proto-oncogene --- p.23, Chapter 1.5.3. --- Genetic Studies in HCC --- p.23, Chapter 1.5.3.1. --- Loss of Heterozygosity (LOH) --- p.25, Chapter 1.5.3.2. --- Comparative Genomic Hybridization (CGH) --- p.26, Chapter 1.5.3.3. --- Array CGH --- p.26, Chapter 1.5.4. --- Large-Scale Genetic Analysis in HCC --- p.27, Chapter CHAPTER2 --- RATIONALE IN THIS STUDY --- p.35, Chapter CHAPTER3 --- MATERIALS AND METHODS, Chapter 3.1. --- Patients and Materials --- p.38, Chapter 3.1.1. --- DNA Extraction --- p.40, Chapter 3.2. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.40, Chapter 3.2.1. --- Microsatellite Markers --- p.41, Chapter 3.2.2. --- Amplification of Target Sequences by PCR --- p.42, Chapter 3.2.2.1. --- 5-end Labeling Primers --- p.42, Chapter 3.2.2.2. --- Amplification of Target Sequences --- p.42, Chapter 3.2.3. --- Denaturing Polyacrylamide Gel --- p.44, Chapter 3.2.3.1. --- Electrophoresis --- p.44, Chapter 3.2.4. --- Detection of Loss of Heterozygosity (LOH) --- p.45, Chapter 3.2.5. --- Duplex PCR Analysis of Homozygous Deletion --- p.45, Chapter 3.3. --- Amplification Analysis by Array-CGH --- p.46, Chapter 3.3.1. --- Nick-Translation --- p.49, Chapter 3.3.2. --- Hybridization --- p.49, Chapter 3.3.3. --- Imaging and Data Analysis --- p.50, Chapter 3.3.4. --- Determination of Normal Range for All Cases --- p.51, Chapter 3.3.5. --- Assessment of Data Quality --- p.51, Chapter 3.4. --- Statistical Analysis --- p.52, Chapter CHAPTER4 --- RESULTS, Chapter 4.1. --- Loss of Heterozygosity Analysis on Chromosome 4q --- p.53, Chapter 4.1.1. --- Region I of Smallest Common Deletion Region --- p.54, Chapter 4.1.2. --- Region II of Smallest Common Deletion Region --- p.54, Chapter 4.2. --- Amplification Analysis by Array-CGH --- p.62, Chapter CHAPTER5 --- DISCUSSION, Chapter 5.1. --- LOH Analysis on Chromosome 4q --- p.73, Chapter 5.1.1. --- LOH of Chromosome 4q in Various Cancers --- p.74, Chapter 5.1.1.1. --- Hepatocellular Carcinomas --- p.74, Chapter 5.1.1.2. --- Other Neoplasia --- p.76, Chapter 5.1.2. --- Functional Studies on Chromosome 4 --- p.76, Chapter 5.1.3. --- Putative Tumor Suppressors on Chromosome 4q --- p.80, Chapter 5.1.3.1. --- Region I (4q27-q28.1) --- p.80, Chapter 5.1.3.1.1. --- MAD2L1 (4q27) --- p.80, Chapter 5.1.3.2. --- Region II (4q35.2) --- p.81, Chapter 5.1.3.2.1. --- INGlL(4q35.1) --- p.81, Chapter 5.1.3.2.2. --- FAT (4q34-q35) --- p.81, Chapter 5.1.3.2.3. --- Caspase 3 (4q35) --- p.82, Chapter 5.1.4. --- Limitation of this Study --- p.83, Chapter 5.1.4.1. --- Markers --- p.83, Chapter 5.1.4.1.1. --- Limitation of the Markers --- p.83, Chapter 5.1.4.1.2. --- Location of the Microsatellite Markers --- p.83, Chapter 5.1.4.2. --- Tissue Samples --- p.84, Chapter 5.1.4.2.1. --- Normal Reference --- p.84, Chapter 5.1.4.2.2. --- Pathologic Characterization --- p.85, Chapter 5.1.5. --- Future Studies --- p.85, Chapter 5.1.5.1. --- Improvement of the Experiment --- p.85, Chapter 5.1.5.2. --- Extension of the Present Study --- p.86, Chapter 5.2. --- Amplification Analysis by Array-CGH --- p.88, Chapter 5.2.1. --- Amplicons Showing Amplification in HCC --- p.89, Chapter 5.2.1.1. --- Locus of 17q23 --- p.89, Chapter 5.2.1.1.1. --- D17S1670 --- p.89, Chapter 5.2.1.1.2. --- RPS6KB1 --- p.91, Chapter 5.2.1.2. --- Locus of 1q25-q31 --- p.92, Chapter 5.2.1.2.1. --- LAMC2 --- p.92, Chapter 5.2.1.3. --- Locus of 3q26.3 --- p.93, Chapter 5.2.1.3.1. --- PIK3CA --- p.93, Chapter 5.2.1.4. --- Locus of 8p22 --- p.94, Chapter 5.2.1.4.1. --- CTSB --- p.94, Chapter 5.2.1.5. --- Locus of 6q22 --- p.95, Chapter 5.2.1.5.1. --- MYB --- p.95, Chapter 5.2.1.6. --- Locus of 20ql3 --- p.96, Chapter 5.2.1.6.1. --- CSE1L --- p.96, Chapter 5.2.1.7. --- Locus of Ip36.2-p35.1 --- p.97, Chapter 5.2.1.7.1. --- FGR --- p.97, Chapter 5.2.1.8. --- Locus of 7q21.1 --- p.98, Chapter 5.2.1.8.1. --- PGY1 --- p.98, Chapter 5.2.2. --- Amplicons Showing Deletion in HCC --- p.99, Chapter 5.2.2.1. --- Loss at 11ql3 and 14q32.3 --- p.99, Chapter 5.2.3. --- Limitation of the Study --- p.100, Chapter 5.2.3.1. --- Samples and Materials --- p.100, Chapter 5.2.4. --- Further Study --- p.101, Chapter 5.2.4.1. --- Confirmation of the Result in Various Levels --- p.101, Chapter 5.2.4.2. --- Assessment of the Significant Losses on Chromosomes 11ql3 and 14ql3 --- p.101, Chapter 5.2.5. --- Application of Microarray in Genetic Studies --- p.102, Chapter 5.2.5.1. --- Deletion Analysis --- p.102, Chapter 5.2.5.2 --- Tissue Microarray --- p.103, Chapter 5.2.5.3. --- cDNA Microarray --- p.103, Chapter chapter6 --- references --- p.104, http://library.cuhk.edu.hk/record=b5891353, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

OBJECTIVE: To review the current knowledge on the genetic alterations involved in the development and progression of Barrett's esophagus-associated neoplastic lesions. SUMMARY BACKGROUND DATA: Barrett's esophagus (BE) is a premalignant condition in which the normal squamous epithelium of the esophagus is replaced by metaplastic columnar epithelium. BE predisposes patients to the development of esophageal adenocarcinoma. Endoscopic surveillance can detect esophageal adenocarcinomas when they are early and curable, but most of the adenocarcinomas are detected at an advanced stage. Despite advances in multimodal therapy, the prognosis for invasive esophageal adenocarcinoma is poor. A better understanding of the molecular evolution of the Barrett's metaplasia to dysplasia to adenocarcinoma sequence may allow improved diagnosis, therapy, and prognosis. METHODS: The authors reviewed data from the published literature to address what is known about the molecular changes thought to be important in the pathogenesis of BE-associated neoplastic lesions. RESULTS: The progression of Barrett's metaplasia to adenocarcinoma is associated with several changes in gene structure, gene expression, and protein structure. Some of the molecular alterations already showed promise as markers for early cancer detection or prognostication. Among these, alterations in the p53 and p16 genes and cell cycle abnormalities or aneuploidy appear to be the most important and well-characterized molecular changes. However, the exact sequence of events is not known, and probably multiple molecular pathways interact and are involved in the progression of BE to adenocarcinoma. CONCLUSIONS: Further research into the molecular biology of BE-associated adenocarcinoma will enhance our understanding of the genetic events critical for the initiation and progression of Barrett's adenocarcinoma, leading to more effective surveillance and treatment.

Alterations in the cellular genome affecting the expression or function of genes controlling cell growth or differentiation are the mechanism underlying the genesis of all cancers. Human cancers often require mutations in genes called proto-oncogenes which mutate to oncogenes (or cancer genes). A subset of proto-oncogenes comprise the RAS signal transduction pathway. Vulval development in the nematode worm C. elegans is controlled by a RAS signal transduction pathway. C. elegans has proven to be a powerful model system for under- standing regulation of the RAS pathway. This research studies two novel components of the RAS pathway in C. elegans, sur-2 and sur-9. A novel gene has been identified that may physically interact with sur-2. In addition, a second novel gene, sur-9, is in the process of being cloned to facilitate further analysis of its function in the RAS signal transduction pathway. These studies will improve our understanding of mitogenic signaling and may eventually lead to the design of novel cancer therapeutic agents which target control proteins in the RAS pathway.

Thyroid tumorigenesis proceeds through the progressive accumulation of alterations in genes involved in the regulation of cell proliferation and differentiation accompanying the acquisition of phenotypic, biological and clinical characteristics of increasing malignancy and dedifferentiation. The molecular alterations specific to thyrocyte carcinogenesis are examined. Ras mutations seem to represent an early occurrence in thyroid tumorigenesis being common to both benign and malignant follicular tumors; they would represent the early mutational events able to enhance the cell proliferation. Subsequent alterations in several genes will probably result in the determination of a follicular or papillary phenotype. In particular, mutational events activating ret, met and trk thyrokinase receptors direct the tumor growth and development towards the papillary type. Progression towards a follicular phenotype would instead occur in two stages: first there is the loss of function of genes on chromosome 11q13 which may direct the tumor cell towards the phenotype of follicular adenoma, second, there is the inactivation of the probable suppressor oncogene on chromosome 3p which might be fundamental in the transition from adenoma to follicular carcinoma. Undifferentiated or anaplastic tumors are characterized by the presence of p53 gene mutations.

In the present study, we analysed 34 de novo diffuse large B cell lymphoma (DLCL) from a population-based lymphoma registry for alterations of the RB1 pathway at the genetic (RB1 and CDK4) and protein (pRb, cyclin D1, cyclin D3, CDK4, and E2F-1) level. The results were correlated with the data from our previous studies of CDKN2A deletion and hypermethylation, other p53 pathway components, p27Kip1 expression, and proliferation, as well as with clinical outcome, including prognosis. We found aberrant pRb expression in four (12%) of 34 DLCLs. One of these had a point mutation in intron 3 10 bp downstream of exon 3 generating a novel splice signal. Seven tumours (21%) showed cyclin D3 overexpression, including all three thyroid lymphomas (P = 0.006). Cyclin D3 overexpression and p16INK4A/pRb aberrations were mutually exclusive, supporting an oncogenic role for cyclin D3 in DLCL. p16INK4A inactivation, cyclin D3 overexpression, or aberrant pRb expression was identified in 18 of 34 DLCLs (53%). Combining these results with our previous p53 pathway studies showed that 82% of the de novo DLCLs had alterations of these pathways, and that both pathways were altered in 13 cases (38%). Low E2F-1 expression was associated with treatment failure (P = 0.020), and multivariate analysis of overall survival identified both low E2F-1 expression (relative risk = 6.9; P = 0.0037) and p16INK4A inactivation (relative risk = 3.3; P = 0.0247) as independent prognostic markers. These data support a role of E2F-1 as tumour suppressor gene in lymphoma and strongly suggest that the RB1 and p53 pathways are important in the development of de novo DLCL. Furthermore, low E2F-1 expression and p16INK4A inactivation may serve as prognostic markers for patients with this type of lymphoma.

In the present study, we analysed 34 de novo diffuse large B cell lymphoma (DLCL) from a population-based lymphoma registry for alterations of the RB1 pathway at the genetic (RB1 and CDK4) and protein (pRb, cyclin D1, cyclin D3, CDK4, and E2F-1) level. The results were correlated with the data from our previous studies of CDKN2A deletion and hypermethylation, other p53 pathway components, p27Kip1 expression, and proliferation, as well as with clinical outcome, including prognosis. We found aberrant pRb expression in four (12%) of 34 DLCLs. One of these had a point mutation in intron 3 10 bp downstream of exon 3 generating a novel splice signal. Seven tumours (21%) showed cyclin D3 overexpression, including all three thyroid lymphomas (P = 0.006). Cyclin D3 overexpression and p16INK4A/pRb aberrations were mutually exclusive, supporting an oncogenic role for cyclin D3 in DLCL. p16INK4A inactivation, cyclin D3 overexpression, or aberrant pRb expression was identified in 18 of 34 DLCLs (53%). Combining these results with our previous p53 pathway studies showed that 82% of the de novo DLCLs had alterations of these pathways, and that both pathways were altered in 13 cases (38%). Low E2F-1 expression was associated with treatment failure (P = 0.020), and multivariate analysis of overall survival identified both low E2F-1 expression (relative risk = 6.9; P = 0.0037) and p16INK4A inactivation (relative risk = 3.3; P = 0.0247) as independent prognostic markers. These data support a role of E2F-1 as tumour suppressor gene in lymphoma and strongly suggest that the RB1 and p53 pathways are important in the development of de novo DLCL. Furthermore, low E2F-1 expression and p16INK4A inactivation may serve as prognostic markers for patients with this type of lymphoma.

Interaction of prolactin (PRL) with its receptor (PRLR) leads to activation of Jak and Src family tyrosine kinases. The PRL/growth hormone/cytokine receptor family conserves a proline-rich sequence in the cytoplasmic juxtamembrane region (Box 1) required for association and subsequent activation of Jaks. In the present work, we studied the mechanisms underlying c-Src kinase activation by PRL and the role that Jak2 plays in this process. PRL addition to chicken embryo fibroblasts (CEF) expressing the rat PRLR long form resulted in activation of c-Src and Jak2 and in tyrosine phosphorylation of the receptor. Receptor phosphorylation was due to associated Jak2, since in cells expressing either a Box 1 mutated PRLR (PRLR(4P-A)), which is unable to interact with Jak2, or a kinase-domain-deleted Jak2 (Jak2Deltak), PRL did not stimulate receptor phosphorylation. Interestingly, addition of PRL to cells expressing PRLR(4P-A) resulted in an activation of c-Src equivalent to that observed with the wild-type receptor. These findings indicate that PRL-mediated stimulation of c-Src was independent of Jak2 activation and of receptor phosphorylation. Our results suggest that PRL-activated Src could send signals to downstream cellular targets independently of Jak2.

Dynamic and complex processes of cell proliferation, differentiation, maturation, apoptosis, and survival maintain homeostasis in bone marrow and perTheral blood. In steady state, the turnover of blood cells is approximately I x 10' cells per day. During stress conditions, such as infections or bleeding, blood cell formation is properly adjusted by the production of terminally differentiated functional cells. Leukemia is a progressive neoplastic disease of the blood cell forming system, in which non-functional blood cells accumulate in the bone marrow, thereby interfering with normal blood cell formation. The lIlain objectives of the experimental work described in this thesis lVere the identification of !lovel prolo-oncogenes or tumor suppressor genes involved ill lIlyeloid leukelllia developlllelll by using retroviral insertionallllutagenesis and the characterization of these novel genes.

The recent advances in the understanding of the pathogenesis of ovarian cancer have been helpful in addressing issues in diagnosis, prognosis and management. The study of ovarian tumours by novel techniques such as immunohistochemistry, fluorescent in situ hybridisation, comparative genomic hybridisation, polymerase chain reaction and new tumour markers have aided the evaluation and application of new concepts into clinical practice. The correlation of novel surrogate tumour specific features with response to treatment and outcome in patients has defined prognostic factors which may allow the future design of tailored therapy based on a molecular profile of the tumour. These have also been used to design new approaches to therapy such as antibody targeting and gene therapy. The delineation of roles of c-erbB2, c-fms and other novel receptor kinases in the pathogenesis of ovarian cancer has led initially to the development of anti-c-erbB2 monoclonal antibody therapy. The discovery of BRCA1 and BRCA2 genes will have an impact in the diagnosis and the prevention of familial ovarian cancer. The important role played by recessive genes such as p53 in cancer has raised the possibility of restoration of gene function by gene therapy. Although the pathological diagnosis of ovarian cancer is still confirmed principally on morphological features, addition of newer investigations will increasingly be useful in addressing difficult diagnostic problems. The increasingly rapid pace of discovery of genes important in disease, makes it imperative that the evaluation of their contribution in the pathogenesis of ovarian cancer is undertaken swiftly, thus improving the overall management of patients and their outcome.

Marginal zone B cell lymphoma (MZBCL) represents a distinct subtype of B cell non-Hodgkin's lymphoma, which has been recently recognized and defined as a disease entity. We investigated 25 cases (18 at primary diagnosis and seven during the course of disease) of MZBCL by comparative genomic hybridization (CGH) and compared these results with cytogenetic, fluorescence in situ hybridization (FISH), and Southern blot data. Twenty of the 25 cases (80%) showed gains (total 49) or losses (total 15) of genetic material. In extranodal, nodal, and splenic MZBCL, material of chromosomes 3 (52% of cases), 18 (32%), X (24%), and 1q (16%) was most frequently gained, whereas losses predominantly involved chromosomes 17 (16%) and 9 (12%). High-level amplifications involving the regions 18q21-23 and 18q21-22, respectively, were detected in two cases. Gains of chromosomes 1q and 8q and losses of chromosome 17 or 17p occurred more frequently in relapsed or progressive lymphomas. For all of the frequently affected chromosomes, CGH allowed narrowing of the relevant subregions including 3q21-23, 3q25-29 and 18q21-23. By Southern blot analysis, the BCL2, BCL6, and CMYC proto-oncogenes were found to be a part of the over-represented regions in two cases, one case, and two cases, respectively, with gains involving 18q, 3q or 8q. In 13 cases, CGH revealed chromosomal imbalances which were not detected by cytogenetic analysis but could be confirmed by FISH or Southern blot analysis in all cases investigated. On the other hand, CGH failed to detect trisomy 3, trisomy 18, and deletion 7q in three cases with a low proportion of tumor cells bearing these abnormalities, as shown by interphase FISH. The characteristic pattern of chromosomal gains and losses detected in this study confirms the distinct nature of MZBCL and may point to chromosomal regions involved in the pathogenesis of these neoplasms.

We used specific antisera and immunohistochemical methods to investigate the subcellular localization and expression of Bcr, Abl, and Bcr-Abl proteins in leukemic cell lines and in fresh human leukemic and normal samples at various stages of myeloid differentiation. Earlier studies of the subcellular localization of transfected murine type IV c-Abl protein in fibroblasts have shown that this molecule resides largely in the nucleus, whereas transforming deletion variants are localized exclusively in the cytoplasm. Here, we demonstrate that the murine type IV c-Abl protein is also found in the nucleus when overexpressed in a mouse hematopoietic cell line. However, in both normal and leukemic human hematopoietic cells, c-Abl is discerned predominantly in the cytoplasm, with nuclear staining present, albeit at a lower level. In contrast, normal endogenous Bcr protein, as well as the aberrant p210BCR-ABL and p190BCR-ABL proteins consistently localize to the cytoplasm in both cell lines and fresh cells. The results with p210BCR-ABL were confirmed in a unique Ph1-positive chronic myelogenous leukemia (CML) cell line, KBM5, which lacks the normal chromosome 9 and hence the normal c-Abl product. Because the p210BCR-ABL protein appears cytoplasmic in both chronic phase and blast crisis CML cells, as does the p190BCR-ABL in Ph1-positive acute leukemia, a change in subcellular location of Bcr-Abl proteins between cytoplasm and nucleus cannot explain the different spectrum of leukemias associated with p210 and p190, nor the transition from the chronic to the acute leukemia phenotype seen in CML. Further analysis of fresh CML and normal hematopoietic bone marrow cells reveals that p210BCR-ABL, as well as the normal Bcr and Abl proteins, are expressed primarily in the early stages of myeloid maturation, and that levels of expression are reduced significantly as the cells mature to polymorphonuclear leukocytes. Similarly, a decrease in Bcr and Abl levels occurs in HL-60 cells induc

Mammalian cells possessing osmosensors have long been described in brain and kidney. The genetic basis of the response to hyperosmotic stress has been well characterized in prokaryotes. In contrast, the genetic response of eukaryotic cells is poorly understood. Therefore we investigated the effect of hypertonic NaCl and sucrose solutions on the transcriptional activation of the immediate-early genes (IEGs) egr-1 and c-fos in isolated ventricular adult rat cardiomyocytes. We observed that even small increases in osmolarity to 315 +/- 5 mosmol/l and 370 +/- 8 mosmol/l by hypertonic NaCl solution resulted in dose-dependent induction of egr-1 (4-and 5-fold) and c-fos (3-and 4-fold), respectively. Hypertonic sucrose solution had the same effect on egr-1 and c-fos mRNA levels while increased sucrose concentration under isotonic conditions had no effect. Cardiomyocytes exposed to hypertonic media did not significantly shrink as shown by a cell length measurement. We conclude that isolated adult cardiomyocytes possess an osmoreceptor mechanism which is able to sense even slight changes in osmolarity and to translate these into a transcriptional response of the myocardial IEG program.

Mammalian cells possessing osmosensors have long been described in brain and kidney. The genetic basis of the response to hyperosmotic stress has been well characterized in prokaryotes. In contrast, the genetic response of eukaryotic cells is poorly understood. Therefore we investigated the effect of hypertonic NaCl and sucrose solutions on the transcriptional activation of the immediate-early genes (IEGs) egr-1 and c-fos in isolated ventricular adult rat cardiomyocytes. We observed that even small increases in osmolarity to 315 +/- 5 mosmol/l and 370 +/- 8 mosmol/l by hypertonic NaCl solution resulted in dose-dependent induction of egr-1 (4-and 5-fold) and c-fos (3-and 4-fold), respectively. Hypertonic sucrose solution had the same effect on egr-1 and c-fos mRNA levels while increased sucrose concentration under isotonic conditions had no effect. Cardiomyocytes exposed to hypertonic media did not significantly shrink as shown by a cell length measurement. We conclude that isolated adult cardiomyocytes possess an osmoreceptor mechanism which is able to sense even slight changes in osmolarity and to translate these into a transcriptional response of the myocardial IEG program.

We used specific antisera and immunohistochemical methods to investigate the subcellular localization and expression of Bcr, Abl, and Bcr-Abl proteins in leukemic cell lines and in fresh human leukemic and normal samples at various stages of myeloid differentiation. Earlier studies of the subcellular localization of transfected murine type IV c-Abl protein in fibroblasts have shown that this molecule resides largely in the nucleus, whereas transforming deletion variants are localized exclusively in the cytoplasm. Here, we demonstrate that the murine type IV c-Abl protein is also found in the nucleus when overexpressed in a mouse hematopoietic cell line. However, in both normal and leukemic human hematopoietic cells, c-Abl is discerned predominantly in the cytoplasm, with nuclear staining present, albeit at a lower level. In contrast, normal endogenous Bcr protein, as well as the aberrant p210BCR-ABL and p190BCR-ABL proteins consistently localize to the cytoplasm in both cell lines and fresh cells. The results with p210BCR-ABL were confirmed in a unique Ph1-positive chronic myelogenous leukemia (CML) cell line, KBM5, which lacks the normal chromosome 9 and hence the normal c-Abl product. Because the p210BCR-ABL protein appears cytoplasmic in both chronic phase and blast crisis CML cells, as does the p190BCR-ABL in Ph1-positive acute leukemia, a change in subcellular location of Bcr-Abl proteins between cytoplasm and nucleus cannot explain the different spectrum of leukemias associated with p210 and p190, nor the transition from the chronic to the acute leukemia phenotype seen in CML. Further analysis of fresh CML and normal hematopoietic bone marrow cells reveals that p210BCR-ABL, as well as the normal Bcr and Abl proteins, are expressed primarily in the early stages of myeloid maturation, and that levels of expression are reduced significantly as the cells mature to polymorphonuclear leukocytes. Similarly, a decrease in Bcr and Abl levels occurs in HL-60 cells induc

Analysis of the regulation of c-jun, jun-B, and c-fos RNA transcript expression was performed in human primary melanocytes and metastatic melanoma cell strains. The medium requirements for human melanocyte in vitro growth are phorbol esters, agents that elevate intracellular cAMP levels, hormones, and growth factors. Cellular jun, jun-B, and c-fos gene expression are known to be affected by growth promoting agents. In primary melanocytes, the expression of c-jun, jun-B, and c-fos RNA transcripts was dependent on the growth-promoting agents present in the medium. Uniformly high c-jun, jun-B, and c-fos RNA transcript levels were observed in melanocytes cultivated in complete medium. Higher levels of c-jun RNA transcripts and low levels of c-fos RNA transcripts were observed in melanocytes cultivated in plain medium. In contrast, a range of c-jun, jun-B, and c-fos RNA transcript levels was detected in metastatic melanoma cell strains cultivated in medium with or without serum. In general, an increase in jun-B and c-fos RNA transcript expression and a decrease in c-jun RNA transcript expression was observed in metastatic melanomas compared to neonatal melanocytes. These data suggest a potential role for c-jun, jun-B, and c-fos genes in the transformation of melanocytes to malignant melanoma.

Three cascades activate thyroid cell proliferation: the EGF-protein tyrosine kinase pathway, the phorbol ester-protein kinase C pathway and the thyrotropin-cyclic AMP pathway. While the first 2 cascades converge early, they remain distinct from the cyclic AMP cascade until very late in G1. The cyclic AMP cascade is characterized by an early and transient expression of c-myc, which may explain why it induces proliferation and differentiation expression. Constitutive activation of this cascade causes growth and hyperfunction, ie, hyperfunctioning adenomas. The various possible defects that could lead to such a constitutive activation are discussed., Journal Article, Review, info:eu-repo/semantics/published

The expressions of the protooncogenes c-jun and jun D have been investigated in dog thyrocytes in a primary culture whose proliferation is stimulated by three distinct intracellular signaling pathways (1) the thyrotropin (TSH) or forskolin-cyclic-AMP-mediated cascade; (2) the protein kinase C pathway activated by diacylglycerol (DAG) and phorbol esters (TPA); (3) a protein tyrosine kinase system activated by epidermal growth factor (EGF). While the first cascade is compatible with the differentiated state of the cell, the two latter pathways induce dedifferentiation. Following the stimulation by TPA or EGF, the expression of c-jun was increased and the expression of jun D was faintly increased. Both expressions are superinduced in the presence of cycloheximide as in mitogenically stimulated fibroblasts but, in the presence of cycloheximide alone, the expressions of c-jun and jun D are clearly unstable with time. This indicates that cycloheximide controls should be included at all time points examined in such experiments. Increasing intracellular concentrations of cyclic-AMP by forskolin or TSH was followed by an inhibition of the expression of c-jun. This inhibition was independent of protein synthesis. Similarly, the TPA or EGF stimulation of c-jun expression was also inhibited by TSH or forskolin, as in fibroblasts in which cyclic-AMP inhibits proliferation. Our results show that the expression of c-jun is not universally correlated with the stimulation of cell proliferation. The stimulation of c-jun expression is not common between the three mitogenic pathways. It thus represents another of the very different responses elicited by the cyclic-AMP cascade as compared to the more studied tyrosine kinase and protein kinase C mitogenic pathways., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Analysis of the regulation of c-jun, jun-B, and c-fos RNA transcript expression was performed in human primary melanocytes and metastatic melanoma cell strains. The medium requirements for human melanocyte in vitro growth are phorbol esters, agents that elevate intracellular cAMP levels, hormones, and growth factors. Cellular jun, jun-B, and c-fos gene expression are known to be affected by growth promoting agents. In primary melanocytes, the expression of c-jun, jun-B, and c-fos RNA transcripts was dependent on the growth-promoting agents present in the medium. Uniformly high c-jun, jun-B, and c-fos RNA transcript levels were observed in melanocytes cultivated in complete medium. Higher levels of c-jun RNA transcripts and low levels of c-fos RNA transcripts were observed in melanocytes cultivated in plain medium. In contrast, a range of c-jun, jun-B, and c-fos RNA transcript levels was detected in metastatic melanoma cell strains cultivated in medium with or without serum. In general, an increase in jun-B and c-fos RNA transcript expression and a decrease in c-jun RNA transcript expression was observed in metastatic melanomas compared to neonatal melanocytes. These data suggest a potential role for c-jun, jun-B, and c-fos genes in the transformation of melanocytes to malignant melanoma.

Overexpression of proto-oncogenes (c-onc) may be involved in the initiation and progression of human neoplasia. We investigated the mRNA expression of the proto-oncogenes c-myc, c-fos, c-neu (erb B-2) and Ha-ras in 12 cutaneous melanocytic lesions (four dermal naevi, seven melanomas; one cutaneous metastatis of melanoma) and in normal skin (five cases) by Northern blot analysis; mRNA expression levels were quantified by densitometry of the specific bands. The expression of c-myc mRNA was higher in naevi than in melanomas, whereas c-fos mRNA expression was significantly higher in melanomas than in naevi. No significant differences were detected in the c-neu and Ha-ras mRNA levels in naevi and melanomas. The expression of c-neu mRNA was higher in normal skin than in the melanocytic lesions examined. Our results suggest that enhanced c-myc and c-fos expression may play an important role in the growth of melanocytic naevi and melanomas. The overexpression of c-fos may be involved in the progression of a particular subset of melanoma. Activation of Ha-ras and the c-neu gene, as determined by mRNA overexpression, does not seem to play a significant role in the progression of cutaneous pigmented lesions. Expression of c-neu may have an important function in the proliferation and/or differentiation of normal cells of the epidermis.

The expressions of the protooncogenes c-jun and jun D have been investigated in dog thyrocytes in a primary culture whose proliferation is stimulated by three distinct intracellular signaling pathways (1) the thyrotropin (TSH) or forskolin-cyclic-AMP-mediated cascade; (2) the protein kinase C pathway activated by diacylglycerol (DAG) and phorbol esters (TPA); (3) a protein tyrosine kinase system activated by epidermal growth factor (EGF). While the first cascade is compatible with the differentiated state of the cell, the two latter pathways induce dedifferentiation. Following the stimulation by TPA or EGF, the expression of c-jun was increased and the expression of jun D was faintly increased. Both expressions are superinduced in the presence of cycloheximide as in mitogenically stimulated fibroblasts but, in the presence of cycloheximide alone, the expressions of c-jun and jun D are clearly unstable with time. This indicates that cycloheximide controls should be included at all time points examined in such experiments. Increasing intracellular concentrations of cyclic-AMP by forskolin or TSH was followed by an inhibition of the expression of c-jun. This inhibition was independent of protein synthesis. Similarly, the TPA or EGF stimulation of c-jun expression was also inhibited by TSH or forskolin, as in fibroblasts in which cyclic-AMP inhibits proliferation. Our results show that the expression of c-jun is not universally correlated with the stimulation of cell proliferation. The stimulation of c-jun expression is not common between the three mitogenic pathways. It thus represents another of the very different responses elicited by the cyclic-AMP cascade as compared to the more studied tyrosine kinase and protein kinase C mitogenic pathways., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Three cascades activate thyroid cell proliferation: the EGF-protein tyrosine kinase pathway, the phorbol ester-protein kinase C pathway and the thyrotropin-cyclic AMP pathway. While the first 2 cascades converge early, they remain distinct from the cyclic AMP cascade until very late in G1. The cyclic AMP cascade is characterized by an early and transient expression of c-myc, which may explain why it induces proliferation and differentiation expression. Constitutive activation of this cascade causes growth and hyperfunction, ie, hyperfunctioning adenomas. The various possible defects that could lead to such a constitutive activation are discussed., Journal Article, Review, info:eu-repo/semantics/published

We reported previously that Ets2 is expressed in normal and transformed macrophages. We show here that the expression of both c-ets-2 mRNA proteins is induced rapidly and transiently in chicken nondividing bone marrow-derived macrophages but not in E26-transformed myeloblasts in response to chicken myelomonocytic growth factor (cMGF), an avian hematopoietic growth factor required for survival, proliferation, and colony formation of avian myeloid cells. c-ets-2 expression is also rapidly induced in chicken bone marrow-derived macrophages, human monocytes, and mouse peritoneal macrophages in response to LPS and/or PKC activators. The rapid induction of Ets2 after treatment of chicken bone marrow-derived macrophages by cMGF is blunted after down-regulation or inactivation of PKC, suggesting a role of PKC in the cMGF-induced signal transduction pathway. Because Ets2 is localized in the nucleus of macrophages and binds to DNA in vitro, the kinetics of its expression suggest a role for Ets2 in the transduction within the nucleus of specific signals received at the cell membrane and involved in securing the survival and/or the development of functional competence of these cells., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

The proliferation of dog thyrocytes in primary culture is stimulated by three distinct intracellular signaling pathways: (1) the thyrotropin or forskolin-cyclic AMP-mediated cascade which is compatible with the differentiated state of the cell; (2) the protein kinase C pathway activated by diacylglycerol and phorbol esters; and (3) a protein tyrosine kinase system activated by epidermal growth factor. The two latter pathways also induce dedifferentiation. The activation of the three cascades induced the expression of the protooncogenes c-fos and c-myc with dose-response curves similar to those for DNA synthesis. After TPA and EGF, the time courses of stimulation of c-fos and c-myc were the same as those for mitogenically stimulated fibroblasts. However, after the cyclic AMP stimulation, c-myc expression was biphasic with an enhancement at 1 h followed by a down-regulation. A similar inhibition by cyclic AMP was also observed on the increased c-myc expression induced by EGF. This down-regulation is suppressed by cycloheximide, which suggests the involvement of a neosynthesized or a labile protein intermediate. The action of cyclic AMP on c-myc mRNA levels could be related to the opposite requirements of the stimulation of both proliferation and differentiation expression by the cyclic AMP pathway in the differentiated thyrocytes., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published