A subgroup of non-small cell lung cancer (NSCLC) cases harbour mutations in classical oncogenes, which can affect therapy response and prognosis. By therapeutically targeting the corresponding proteins with inhibitory drugs, the clinical outcome for these lung cancer patients may be improved. One of these oncogenes is the phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) which encodes the catalytic subunit of the phosphatidylinositol 3 kinase (PI3K). PIK3CA is a central regulator in the PI3K/Akt/mTOR pathway, which controls cell growth and apoptosis. Mutations in the PIK3CA gene are considered to up-regulate the kinase activity in tumour cells and through that dysregulate fundamental cellular processes. PI3K inhibitors are currently tested in clinical trials and present a promising therapy option in lung cancer patients. In this study, a pyrosequencing assay for detection of PIK3CA mutations in tumours from patients with NSCLC was established. The three "hot-spot" codons 542, 545 and 1047 of the PIK3CA gene were analysed. The sensitivity of this assay was determined to the presence of 5 % of mutant alleles. In agreement with previous reports, three of the 60 lung cancer cases revealed PIK3CA mutations (5 %). All mutations occurred in exon 9 codon 542 or 545. In line with previous reports, two of the three samples harboured concurrent mutation in the EGFR or KRAS gene. The established pyrosequencing analysis for PI3KCA mutations provides a reliable and cost-effective assay for clinical diagnostics. The determination of the PI3KCA mutation status may help to distinguish patients for treatment targeting the PI3K pathway.

Leung Chun-on, Paul. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 149-167). / Content Page / Acknowledgments --- p.7 / Chapter / Chapter Chapter1 --- Introduction --- p.8 / Chapter Chapter2 --- Literature Review --- p.13 / Chapter 2.1 --- Anatomy of the cervix --- p.13 / Chapter 2.2 --- Classification --- p.14 / Chapter 2.2.1 --- Cervical intraepithelial neoplasia (CIN) --- p.14 / Chapter 2.2.2 --- Cervical cancer --- p.17 / Chapter 2.2.3 --- Incidence and screening --- p.21 / Chapter 2.2.4 --- Etiology / Chapter 2.2.4.1 --- Sexual and reproductive factors --- p.23 / Chapter 2.2.4.2 --- Smoking as a risk factor --- p.23 / Chapter 2.2.4.3 --- Male partner contribution --- p.24 / Chapter 2.2.4.4 --- Human papillomaviruses and cervical cancer --- p.24 / Chapter 2.2.4.5 --- Oral contraceptive pills --- p.27 / Chapter 2.2.4.6 --- Oncogenes and tumour suppresser genes --- p.28 / Chapter 2.2.4.7 --- Oncogenes and cervical cancer --- p.35 / Chapter 2.3 --- Immunohistochemical technique in cancer study / Chapter 2.3.1 --- Principle of immunostaining --- p.39 / Chapter 2.3.2 --- Fixation --- p.40 / Chapter 2.3.3 --- Section preparation --- p.41 / Chapter 2.3.4 --- The choice of antibodies --- p.41 / Chapter 2.3.5 --- Enzyme labels --- p.42 / Chapter 2.3.6 --- Blocking endogenous enzymes --- p.43 / Chapter 2.3.7 --- Blocking background staining --- p.43 / Chapter 2.3.8 --- Dilution preparation --- p.44 / Chapter 2.3.9 --- The Avidin-Biotin technique --- p.44 / Chapter 2.3.10 --- Control --- p.47 / Chapter 2.3.11 --- Antigen retrieval --- p.47 / Chapter 2.3.12 --- Cell counting and scoring --- p.49 / Chapter 2.4 --- The application of Polymerase Chain Reaction Single-Strand Conformation Polymorphism(PCR-SSCP) in cancer study --- p.52 / Chapter Chapter3 --- Materials and Methods --- p.56 / Chapter 3.1 --- Materials --- p.56 / Chapter 3.2 --- Methods --- p.61 / Chapter 3.2.1 --- Specimens collection --- p.61 / Chapter 3.2.2 --- Antibodies preparation --- p.63 / Chapter 3.2.3 --- Immunohistochemical staining and antigen retrieval procedures --- p.63 / Chapter 3.2.4 --- Cell counting and scoring --- p.68 / Chapter 3.2.5 --- PCR-SSCP analysis for myc gene mutation --- p.70 / Chapter 3.2.5.1 --- DNA extraction --- p.70 / Chapter 3.2.5.2 --- PCR --- p.72 / Chapter 3.2.5.3 --- Preparing the single strand DNA --- p.73 / Chapter 3.2.5.4 --- Electrophoresis --- p.73 / Chapter 3.2.5.5 --- Gel drying and scanning --- p.77 / Chapter 3.2.6 --- Statistical analysis --- p.77 / Chapter Chapter 4 --- Result --- p.78 / Chapter Chapter 5 --- Discussion --- p.126 / Chapter Chapter 6 --- Conclusions --- p.144 / Reference --- p.148

Tsang Sup-Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 176-185). / Abstracts in English and Chinese. / Acknowledgments --- p.1 / Abstract --- p.2 / 摘要 --- p.4 / List of Abbreviation --- p.6 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Isolation and activation of mas oncogene --- p.11 / Chapter 1.2 --- Amino acid sequence of mas oncogene --- p.14 / Chapter 1.3 --- Expression of mas oncogene --- p.18 / Chapter 1.4 --- Possible physiological role of mas oncogene --- p.20 / Chapter 1.5 --- Gene related to mas family --- p.23 / Chapter 1.6 --- Aims of study --- p.26 / Chapter Chapter 2 --- Over-expression of mas oncogene / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.2 --- Materials and Methods --- p.29 / Chapter 2.2.1 --- Materials --- p.30 / Chapter 2.2.1.1 --- Chemicals --- p.30 / Chapter 2.2.1.2 --- Enzyme --- p.30 / Chapter 2.2.1.3 --- DNA Purification Kit --- p.31 / Chapter 2.2.1.4 --- Others --- p.31 / Chapter 2.2.2 --- Methods --- p.31 / Chapter 2.2.2.1 --- Strategy of construct preparation --- p.31 / Chapter 2.2.2.2 --- "Preparation of linearized vector, pFRSV" --- p.32 / Chapter 2.2.2.2.1 --- Cloning of vectors --- p.32 / Chapter 2.2.2.2.2 --- Restriction enzyme digestion and DNA dephosphorylation --- p.34 / Chapter 2.2.2.2.3 --- DNA purification by agarose gel electro-elution --- p.34 / Chapter 2.2.2.3 --- Preparation of pFRSV/mas construct --- p.35 / Chapter 2.2.2.3.1 --- PCR amplification --- p.35 / Chapter 2.2.2.3.2 --- Restriction enzyme digestion --- p.36 / Chapter 2.2.2.4 --- Ligation and analysis --- p.37 / Chapter 2.2.2.5 --- Purification of DNA by cesium chloride --- p.38 / Chapter 2.2.2.5.1 --- Large-scale bacterial culturing --- p.38 / Chapter 2.2.2.5.2 --- Ethanol precipitation --- p.39 / Chapter 2.2.2.5.3 --- Cesium chloride purification --- p.39 / Chapter 2.2.2.5.4 --- Removal of DNA dye by dialysis and ethanol precipitation --- p.40 / Chapter 2.2.2.6 --- Transfection by electroporation --- p.41 / Chapter 2.2.2.7 --- Screening for the stably transfected cells --- p.42 / Chapter 2.2.2.8 --- RT-PCR analysis of the mas transfectant --- p.43 / Chapter 2.2.2.8.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.43 / Chapter 2.2.2.8.2 --- Reverse transcription of the total RNA into cDNA --- p.44 / Chapter 2.2.2.8.3 --- Analysis of the transfected mas expression by PCR --- p.44 / Chapter 2.2.2.8.4 --- Analysis of the transfected DHFR expression by PCR --- p.45 / Chapter 2.2.2.8.5 --- Analysis of endogenous GAPDH expression by PCR --- p.46 / Chapter 2.2.2.9 --- Amplification of mas transgene by using methotrexate --- p.47 / Chapter 2.2.2.9.1 --- Amplification by low dosage MTX treatment --- p.47 / Chapter 2.2.2.9.2 --- Amplification by high dosage MTX treatment --- p.49 / Chapter 2.2.2.10 --- Southern blot analysis --- p.50 / Chapter 2.2.2.10.1 --- Preparation of DIG-labelled mas probe --- p.51 / Chapter 2.2.2.10.2 --- Preparation of DIG-labelled DHFR probe --- p.51 / Chapter 2.2.2.10.3 --- Preparation of DIG-labelled GAPDH probe --- p.52 / Chapter 2.2.2.10.4 --- Isolation of Genomic DNA from the mas transfectants by DNAzol® Reagent / Chapter 2.2.2.10.5 --- Enzymatic restriction of genomic DNA and Gel electrophoresis --- p.54 / Chapter 2.2.2.10.6 --- DNA transferring to positive charged Nylon membrane --- p.54 / Chapter 2.2.2.10.7 --- Pre-hybridization and hybridization --- p.56 / Chapter 2.2.2.10.8 --- Post-hybridization washing and blocking --- p.56 / Chapter 2.2.2.10.9 --- Detection --- p.57 / Chapter 2.2.2.11 --- Northern blot analysis --- p.57 / Chapter 2.2.2.11.1 --- Preparation of the agarose gel containing formaldehyde --- p.58 / Chapter 2.2.2.11.2 --- Preparation of the RNA sample --- p.58 / Chapter 2.2.2.11.3 --- Gel electrophoresis and transferring --- p.59 / Chapter 2.2.2.11.5 --- Pre-hybridization and hybridization --- p.60 / Chapter 2.2.2.11.4 --- Post-hybridization washing and blocking --- p.60 / Chapter 2.2.2.11.6 --- Detection --- p.61 / Chapter 2.2.2.11.7 --- Stripping and rehybridization --- p.61 / Chapter 2.3 --- Results --- p.62 / Chapter 2.3.1 --- RT-PCR analysis of gene expression in the stably transfectant --- p.62 / Chapter 2.3.2 --- Morphology of the mas trasnfectant --- p.64 / Chapter 2.3.3 --- Determination of mas gene copy number by Southern blot analysis in the mas transfectants --- p.66 / Chapter 2.3.4 --- Northern blot analysis of the transcriptional level of mas transcriptsin mas transfectants --- p.76 / Chapter 2.4 --- Discussion --- p.87 / Chapter Chapter 3 --- In vivo study of physiological effect of over-expression of mas / Chapter 3.1 --- Introduction --- p.92 / Chapter 3.2 --- Materials and Methods --- p.93 / Chapter 3.2.1 --- Materials --- p.93 / Chapter 3.2.2 --- Methods --- p.93 / Chapter 3.2.2.1 --- Cell culture --- p.93 / Chapter 3.2.2.2 --- Subcutaneous injection of nude mice --- p.94 / Chapter 3.2.2.3 --- Isolation of the total RNA from the tumor tissues --- p.95 / Chapter 3.2.2.4 --- Northern blot analysis --- p.96 / Chapter 3.3 --- Results --- p.96 / Chapter 3.3.1 --- Tumorgenicity assay of mas oncogene in nude mice --- p.96 / Chapter 3.3.2 --- Northern blot analysis of mas expression in the tumor tissues --- p.103 / Chapter 3.4 --- Discussion --- p.109 / Chapter Chapter 4 --- Fluorescent differential display analysis of mas transfectants / Chapter 4.1 --- Introduction --- p.111 / Chapter 4.2 --- Materials and Methods --- p.112 / Chapter 4.2.1 --- Materials --- p.112 / Chapter 4.2.1.1 --- Chemicals --- p.112 / Chapter 4.2.1.2 --- Enzyme --- p.113 / Chapter 4.2.1.3 --- Kits --- p.113 / Chapter 4.2.1.4 --- Others --- p.114 / Chapter 4.2.2 --- Methods --- p.114 / Chapter 4.2.2.1 --- Isolation of the total RNA from the mas transfectants by TRIzol ® Reagent --- p.114 / Chapter 4.2.2.2 --- DNase I treatment --- p.115 / Chapter 4.2.2.3 --- Reverse transcription (RT) and non-fluorescent PCR --- p.116 / Chapter 4.2.2.4 --- Reverse transcription and fluorescent differential display-PCR --- p.118 / Chapter 4.2.2.5 --- High resolution fluorescent differential display (Fluoro DD) gel --- p.118 / Chapter 4.2.2.6 --- Gel band excision of differentially expressed cDNA fragments --- p.120 / Chapter 4.2.2.7 --- Gel band reamplification --- p.120 / Chapter 4.2.2.8 --- Subcloning of reamplified cDNA fragments --- p.121 / Chapter 4.2.2.9 --- Purification of plasmid DNA from recombinant clones for reverse dot blot analysis --- p.122 / Chapter 4.2.2.10 --- Reverse dot blot analysis --- p.123 / Chapter 4.2.2.10.1 --- Preparation of cDNA dot blot --- p.123 / Chapter 4.2.2.10.2 --- Preparation of DIG-labeled cDNA library probes --- p.124 / Chapter 4.2.2.10.3 --- Hybridization --- p.126 / Chapter 4.2.2.11 --- Northern blot analysis --- p.127 / Chapter 4.3 --- Results --- p.128 / Chapter 4.3.1 --- Fluorescent differential display (FluoroDD) --- p.128 / Chapter 4.3.2 --- Reverse dot blot analysis --- p.135 / Chapter 4.3.3 --- DNA sequencing analysis of the clones --- p.141 / Chapter 4.3.4 --- Confirmation of differential display pattern of the subclones by Northern blot analysis --- p.160 / Chapter 4.4 --- Discussion --- p.166 / Chapter Chapter 5 --- General Discussion / Chapter 5.1 --- General model for mos-induced tumor formation --- p.169 / Chapter 5.2 --- Future aspect --- p.174 / References --- p.176 / Appendix I Buffer composition --- p.186 / Appendix II Sequences of fluoroDD TMR-Anchored primers and arbitrary primers --- p.190

<p>The development of cancer is a process by which an accumulation of genetic changes leads to uncontrolled replication of cells. Since the process of mutation is random, the set of alterations that occur and accumulate during tumorigenesis in one individual is different from that of another. These genetic differences drive tumor heterogeneity. One of the first technologies used to explore genome-wide heterogeneity was the microarray, which can be used to measure the expression of tens of thousands of genes. By exploring differences in expression of not just single genes, but groups of genes that may be altered in one set of tumors compared to another, researchers were able to classify subtypes of cancer that had relevance in disease aggressiveness, treatment, and prognosis. Furthermore, by looking at genome-wide patterns of expression, it is possible to identify specific oncogenic pathways that are activated and critical in driving tumor cell survival, growth, or metastasis. My research utilizes the patterns of expression derived from microarray analyses to study tumor heterogeneity, particularly in response to targeted cancer therapy, and mechanisms of cell death following oncogenic deregulation.</p><p>One of the cancer types that has been explored through expression array analysis is B-cell lymphoma. Human aggressive B-cell non-Hodgkin lymphomas (NHL) encompass the continuum between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), and display considerable clinical and biologic heterogeneity, most notably related to therapy response. We previously showed that lymphomas arising in the E&mu-Myc transgenic mouse are heterogeneous, mirroring genomic differences between BL and DLBCL. Given the clinical heterogeneity in NHL and the need to develop strategies to match therapeutics with discrete forms of disease, we investigated the extent to which genomic variation in the E&mu-Myc model predicts response to therapy. We used genomic analyses to classify E&mu-Myc lymphomas, link E&mu-Myc lymphomas with NHL subtypes, and identify lymphomas with predicted resistance to conventional and NF-&kappaB targeted therapies. Experimental evaluation of these predictions links genomic profiles with distinct outcomes to conventional and targeted therapies in the E&mu-Myc model, and establishes a framework to test novel targeted therapies or combination therapies in specific genomically-defined lymphoma subgroups. In turn, this will rationally inform the design of new treatment options for aggressive human NHL.</p><p>The second aspect of my thesis looks at the mechanisms of apoptosis following oncogene deregulation. The Rb-E2F pathway is a critical oncogenic pathway that is frequently mutated in cancers. Alterations in the pathway affect genome-wide expression in the cell, which in turn lead to deregulation of the cell cycle. The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors that cooperate with E2F to mediate the specificity of E2F function. In this work, we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance. </p><p>Taken together, these studies highlight the importance and power of analyzing genome-wide patterns of expression in investigating cancer heterogeneity, its ability to help predict treatment response, and its role in discovering the mechanisms behind the consequences of gene deregulation.</p> / Dissertation

The oncogene Ahi-1 was recently identified through provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in several leukemic cells lines, particularly in cutaneous T-cell lymphoma (CTCL) cell lines (Hut 78 and Hut 102). Hut 78 is derived from a patient with Sezary syndrome, a common leukemic variant of the human CTCL mycosis fungoides. Aberrant expression of AHI-1 mRNA and protein has been found in CD4⁺CD7⁻ leukemic Sezary cells from patients with Sezary syndrome. Moreover, stable suppression of AHI-1 using retroviral-mediated RNA interference in Hut 78 cells inhibits their transforming activity in vitro and in vivo. In an effort to identify genes involved in AHI-1-mediated leukemic transformation in CTCL, microarray analysis was performed to compare six RNA samples from AHI-1 suppressed Hut 78/sh4 cells to five samples from Hut 78 control cells. Limma and dChip analyses identified 218 and 95 differentially expressed genes, respectively, using a fold change criteria of > or < 2 and a p-value threshold of ≤ 0.01. After evaluation of both analyses, 21 genes were selected based upon interesting structural and functional information, specificity to hematopoietic cells or T-cells, and previous connections to cancer. Expression patterns of these 21 genes were validated by qRT-PCR with p-values < 0.05 ranging from 1.97 x 10⁻¹⁰ to 6.55 x 10⁻³, with the exception of BRDG1 at 5.88 x 10⁻². The observed up-regulation of both BIN1 and HCK in AHI-1 suppressed Hut 78/sh4 cells as compared to control cells further confirmed at the protein level. The tumor suppressor BIN1 is known to physically interact with c-MYC, which also exhibits differential protein expression in these cells. Characterization of BIN1 identified 4 isoforms all of which contain exon 10 and demonstrate alternative splicing of exons 12A and 13. Additionally, qRT-PCR results from primary Sezary samples indicate there is clinical significance in the expression changes detected for BIN1, HCK, REPS2, BRDG1, NKG7 and SPIB. These findings identify several new differentially expressed genes that may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells.

Human papillomavirus (HPV) infections play a crucial role in human carcinogenesis. Greater than 96% of all cervical carcinomas are positive for high-risk HPV infections; especially types 16 and 18. High-risk HPV onco-proteins, E6 and E7, are consistently expressed in such cancers and function by inactivating p53 and pRb tumor suppressors, respectively. The presence of high-risk HPVs is also correlated with anogenital cancers. In this study, we examined the effect of high-risk HPV type 16 E6 and E7 oncoproteins in two normal human colorectal epithelial cell lines, NCE1 and NCE5. We report that the expression of E6/E7 proteins, alone, induced cellular transformation of both cell lines; consequently, NCE1-E6/E7 and NCE5-E6/E7 form colonies in soft agar with respect to their wild type cells. This is accompanied by cell cycle deregulation, as is demonstrated by the over-expression of cyclin dependant kinases (cdks) and their respective cyclins. Furthermore, we demonstrate that E6/E7 oncoprotein transduction induces migration of colorectal epithelial cells. More still, well analyzed Id gene expression, a family member of the helix-loop-helix (HLH) transcription factors involved in the regulation of cell invasion and metastasis of human cancer cells. In parallel, using tissue microarray analysis we found that the four members of the Id protein family are correlated with the presence of HPV type 16 and 18 in human colon cancer tissues. Our data suggests that high-risk HPV infections are sufficient to induce cellular transformation of normal human colorectal cells, in vitro. Furthermore, the correlation with the Id family of proteins may present a novel set of markers associated with HPV induced colorectal carcinogenesis. Our results may suggest a new approach to detect and prevent colorectal cancer.

The oncogene Ahi-1 was recently identified through provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in several leukemic cells lines, particularly in cutaneous T-cell lymphoma (CTCL) cell lines (Hut 78 and Hut 102). Hut 78 is derived from a patient with Sezary syndrome, a common leukemic variant of the human CTCL mycosis fungoides. Aberrant expression of AHI-1 mRNA and protein has been found in CD4⁺CD7⁻ leukemic Sezary cells from patients with Sezary syndrome. Moreover, stable suppression of AHI-1 using retroviral-mediated RNA interference in Hut 78 cells inhibits their transforming activity in vitro and in vivo. In an effort to identify genes involved in AHI-1-mediated leukemic transformation in CTCL, microarray analysis was performed to compare six RNA samples from AHI-1 suppressed Hut 78/sh4 cells to five samples from Hut 78 control cells. Limma and dChip analyses identified 218 and 95 differentially expressed genes, respectively, using a fold change criteria of > or < 2 and a p-value threshold of ≤ 0.01. After evaluation of both analyses, 21 genes were selected based upon interesting structural and functional information, specificity to hematopoietic cells or T-cells, and previous connections to cancer. Expression patterns of these 21 genes were validated by qRT-PCR with p-values < 0.05 ranging from 1.97 x 10⁻¹⁰ to 6.55 x 10⁻³, with the exception of BRDG1 at 5.88 x 10⁻². The observed up-regulation of both BIN1 and HCK in AHI-1 suppressed Hut 78/sh4 cells as compared to control cells further confirmed at the protein level. The tumor suppressor BIN1 is known to physically interact with c-MYC, which also exhibits differential protein expression in these cells. Characterization of BIN1 identified 4 isoforms all of which contain exon 10 and demonstrate alternative splicing of exons 12A and 13. Additionally, qRT-PCR results from primary Sezary samples indicate there is clinical significance in the expression changes detected for BIN1, HCK, REPS2, BRDG1, NKG7 and SPIB. These findings identify several new differentially expressed genes that may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells.

Thesis (Ph. D.)--Cornell University, January, 1989. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Virginia Commonwealth University, 2006. / Prepared for: Dept. of Biochemistry. Bibliography: leaves 83-87. Also available online.

Abstract Adhesive interactions between cells and extracellular matrix proteins play a vital role in biological processes such as cell proliferation, differentiation and survival. Integrins comprise a major family of cell surface receptors that mediate these interactions. Integrin engagement triggers adhesion-dependent intracellular signalling cascades that include the phosphorylation of tyrosines in intracellular signalling proteins. Integrin-dependent signals act in concert with signals from growth factors and other signalling receptors. The objective of this thesis was to study how cell adhesion and growth factors interact with intracellular components to regulate cell behavior in normal and transformed cells. One of the main proteins phosphorylated following integrin ligation in several different cell types is the docking protein p130Cas (Cas), which is tyrosine phosphorylated after stimulation of cells with low concentrations of epidermal growth factor (EGF). Tyrosine-phosphorylated Cas associates with an adapter protein c-Crk, the main binding protein for Cas, suggesting a novel role for EGF in Cas signalling. The interaction of cells with a variety of agonists such as growth factors and integrin ligation results in stimulation of mitogen-activated protein kinases (MAPKs), which control the expression of genes important for many cell functions. Expression of Cas and Crk induces activation of C-Jun N-terminal kinases (JNKs), which are members of MAPK family. JNK activation induced by integrin ligand binding is blocked by the expression of a dominant-negative mutant of Cas or Crk demonstrating an important role for the Cas-Crk complex in integrin-mediated JNK activation. The proto-oncogene product p120Cbl (Cbl) was identified as the main tyrosine-phosphorylated protein following integrin ligation in hematopoietic cells of myeloid lineage. Tyrosine-phosphorylated Cbl interacts with and activates other signalling proteins, such as Src tyrosine kinase and phosphatidylinositol 3"-kinase (PI 3-kinase), thereby mediating adhesion-dependent signals in hematopoietic cells. Unlike the cellular Cbl, the transforming mutants of Cbl were tyrosine-phosphorylated in an adhesion-independent manner and interacted with and activated signalling molecules both in suspended and in adherent cells. Further, the oncogenic forms of Cbl induced anchorage-independent but serum-dependent proliferation of cells. These results support the view that transformation by Cbl results from constitutive activation of integrin-dependent rather than growth factor-dependent signalling events.