Undergraduate Research Assistants PDF

I have openings for undergraduate research assistants in my lab open to sophomores and juniors must be able to work up to 20 hours per week-at least a one year committment $15/hour Let me know if you are interested “If you live long enough you will die of cancer” Fundamental pro...

I have openings for undergraduate research assistants in my lab open to sophomores and juniors must be able to work up to 20 hours per week-at least a one year committment $15/hour Let me know if you are interested “If you live long enough you will die of cancer” Fundamental properties of cancer Cancer cells share two fundamental properties: – unregulated cell proliferation – metastatic spread  Cancer is a genetic disease Genomic alterations that are associated with cancer range from single-nucleotide substitutions to large-scale chromosomal rearrangements, amplifications, and deletions.  Development of Cancer Most cancers derive from a single abnormal cell Evidence from X- inactivation mosaics demonstrates the monoclonal origin of cancer. “Primary” (normal) cells versus “transformed” cells Primary cells can be grown in culture – require growth factors – survive a limited number of cell divisions Features of typically adherent primary cells (e.g., fibroblasts, endothelial cells, smooth muscle cells) are lost upon transformation. These include: – anchorage dependence – serum dependence – density-dependent growth inhibition (contact- inhibition).  Cancer is usually a multistep process Cancer is a multistep process requiring multiple mutations. Age-related cancer is an indication that cancer develops from the accumulation of several mutagenic events.  Multi-hit theory of oncogenesis It was believed that appearance of tumors in vivo involves more than one genetic defect. This was demonstrated using transgenic mice carrying oncogenes. Tumor formation in mice carrying one or multiple oncogenes  Tumor progression involves successive rounds of mutation followed by natural selection. Clonal evolution: A tumor develops through repeated rounds of mutation and proliferation, giving rise eventually to a clone of fully malignant cancer cells. At each step, a single cell undergoes a mutation that enhances cell proliferation, so that its progeny become the dominant clone in the tumor. Proliferation of this clone then hastens occurrence of the next step of tumor progression by increasing the size of the cell population at risk of undergoing an additional mutation.  Genomic instability The genomic instability in cancer cells manifests itself in gross defects such as: – translocations – aneuploidy – chromosome loss – DNA amplification – chromosomal deletions  Genetic instability and tumor progression Cells that maintain an “optimum” level of genetic instability may be the most successful in tumor formation.  Epigenetic changes Epigenetic changes (factors that affect gene expression in a heritable way but do not alter the nucleotide sequence of DNA) such as DNA methylation and histone acetylation (enhances transcription) modify gene expression, potentially leading to tumor progression. For example, the DNA repair genes MLH1 and BRCA1 are transcriptionally silenced in certain cancer cells.  Epigenetic changes contribute to tumor progression Epigenetic changes can also create selective advantages to tumor cells. Many mutations that make cells cancerous alter the proteins that determine chromatin structures. Tumor progression includes the development of the ability to metastasize. Steps towards metastasis Loss of contact inhibition – Cancer cells, unlike most normal cells, usually continue to grow and pile up on top of one another after they have formed a confluent monolayer. Loss of adherence due to : – cytoskeletal changes – changes in expression of adhesion molecules – increased expression of proteases resulting in degradation of extracellular matrix. Changes also include the expression of angiogenic factors such as vascular Steps in the process of metastasis  The tumor microenvironment influences cancer development Tumors consist of many cell types, in addition to the cancer cells, including fibroblasts, vascular epithelial cells, smooth muscle cells, and leukocytes. There is cross-talk between the cancer cells and its associated stromal cells. Cancer cells secrete cytokines and growth factors that act on stromal cells directing them to create a favorable environment for the tumor.  Tumor cells evade the immune system and recruit cells of the immune system to support their growth and metastasis Tumors evade elimination by the immune system by: – downregulating major histocompatibility proteins – producing factors that suppress immune function such as TGFbeta and Fas Ligand. Tumors secrete chemokines that recruit leukocytes that provide support for the tumor. – Tumor associated macrophages (TAMs) produce growth factors and other proteins that support tumor growth  Top 9 features that make a cancer cell successful 1. They disregard the external and internal signals that regulate cell proliferation. 2. They can avoid apoptosis. 3. They circumvent programmed limitation to proliferation by escaping replicative senescence (stabilize their telomeres). 4. They induce help from normal cells in their local environment 5. They induce angiogenesis. 6. They are genetically unstable. 7. They escape from their home tissues (they are invasive). 8. They survive and can proliferate in foreign sites (they metastasize). 9. They evade the immune system.  Inducers of Cancer Tumor initiators: agents which cause DNA damage Tumor promoters: agents that do not directly damage DNA, but act to promote tumor formation  Tumor Initiators damage DNA – chemical carcinogens aromatic hydrocarbons nitrosamines alkylating agents – viruses – radiation: UV radiation, ionizing radiation (gamma rays and alpha particles)  Tumor promoters promote cell proliferation Substances that are mitogenic can act as tumor promoters. – Example: phorbol esters, which act as activators of PKC. – Inflammation can act as a tumor promoter The tumor promoter expands the population of mutant cells, thereby increasing the probability of tumor progression by further genetic change. Genetic changes resulting in oncogenesis Oncogenic mutations include those that result in both gain of function and loss of function  Cancers involving DNA repair A number of inherited cancers are caused by defects in genes that control DNA repair, including: – xeroderma pigmentosum…defective in nucleotide excision repair – hereditary nonpolyposis colorectal cancer (HNPCC)…Mismatch repair enzymes  A translocation results in Chronic Myelogenous Leukemia (CML) translocation between Philadelphia chromosome chromosome 22 and 9 joins the bcr gene (break point cluster region) on chromosome 22 to the Abl gene from chromosome 9 resulting fusion protein has the amino terminus of the Bcr protein joined to the carboxyl terminus of the Abl tyrosine protein kinase. the Abl kinase domain becomes inappropriately active, driving excessive proliferation of a clone of hematopoietic cells in the bone marrow.  The Bcr-Abl protein as a target for therapeutic intervention Since the mutation resulting in CML was known and the Bcr-Abl protein had been characterized, this allowed for the design of drugs which specifically block the function of the Bcr-Abl kinase. A drug called STI-571, now called Gleevec, inhibits Bcr-Abl activity and is being used to treat CML patients.  Gleevec (STI-571) blocks the activity of Bcr-Abl protein Gleevec sits in the ATP-binding pocket of the tyrosine kinase domain of Bcr-Abl and thereby prevents Bcr-Abl from transferring a phosphate group from ATP onto a tyrosine residue in a substrate protein. This blocks onward transmission of a signal for cell proliferation and survival. Cancer Cells Contain Genetic Defects Affecting Cell-Cycle Regulation Growth and differentiation of cells are strictly regulated. In cancer cells, many of the genes that control these functions are mutated or aberrantly expressed, leading to uncontrolled cell proliferation.  G0 Cells that stop proliferating enter G0, in which they just hang out (do not grow or divide but are metabolically active). Cancer cells are unable to enter G0 and cycle continuously.  Apoptosis Cells halt progress through the cell cycle if DNA replication, repair, or chromosome assembly are aberrant. If DNA damage is so severe that repair is impossible, the cell may initiate apoptosis, or programmed cell death. A series of proteases called caspases are responsible for initiating apoptosis and for digesting intracellular components.  Proto-oncogenes Proto-oncogenes are genes whose products promote cell growth and division. These genes encode: – transcription factors that stimulate expression of other genes – signal transduction molecules that stimulate cell division – cell-cycle regulators that move through the cell cycle – An oncogene is a proto-oncogene that is mutated or aberrantly expressed (gain of function alteration) and  Tumor suppressor genes The products of tumor suppressor genes normally regulate cell cycle checkpoints and initiate the process of apoptosis. When tumor suppressor genes are mutated or inactivated, cells are unable to respond normally to cell cycle checkpoints or are unable to undergo apoptosis if DNA damage is extensive.  Mutations in p53 Mutations in p53 or other components of the p53 pathway are mutated in nearly all human cancers. Reminder: p53 acts by holding cells in G1 arrest (through the induction of the CKI, p21) to allow for DNA repair. Mutations in p53 allows mutant cells to continue through cell cycle. Mutations in p53 allow escape from apoptosis. Mutations in p53 allow for further mutations to occur as the cells divide.  Absence of a functional p53 can lead to chromosome abnormalities, gene amplification and gene loss. Scenario: Accidental DNA damage occurs in a cell that lacks functional p53 protein. Instead of halting at the p53-dependent checkpoint in the division cycle, the p53-defective cell enters S phase. Chromosomes carrying a duplication and lacking a telomere may be generated - allowing repeated rounds of replication, chromatid fusion, and unequal breakage which can can increase the number of copies of the duplicated region still further. Called the “breakage-fusion-bridge cycle”  The breakage-fusion-bridge cycle Selection in favor of cells with increased numbers of copies of a gene in the affected chromosomal region will lead to mutants in which the gene is amplified to a high copy number. The chromosomal disorder can also lead to loss of genes, with selection in favor of cells that have lost tumor suppressors. How shortened telomeres may lead to chromosomal instability and cancer. Normal Cancer  Role of telomeres in cancer Most somatic cells types can undergo a limited number of divisions in cell culture. – this phenomenon is known as replicative cell senescence – caused by changes in telomeres, the repetitive DNA sequences and associated proteins at the ends of chromosomes. Telomeric sequences are not replicated like the rest of the DNA in a cell, but are synthesized by the enzyme telomerase. Most cell types do not express telomerase. Therefore, the telomeres become shorter with each cell division. Eventually DNA damage occurs at the chromosome ends which results in p53 activation of cell cycle arrest. Lack of telomerase in somatic cells protects against unrestricted proliferation. – Could this inadvertently cause cancer??? Proto-oncogene-ras. Mutations that cause Ras not to hyrdrolyze GTP cause the cell to become cancerous  RB1 Loss or mutation of both alleles of the RB1 tumor suppressor gene contributes to the development of many cancers due to unregulated progression through the cell cycle. In familial retinoblastoma, a mutated RB1 allele is inherited. Sporadic retinoblastoma requires two independent mutational events of RB1 within the same cell.  Cancer Cells Metastasize, Invading Other Tissues To metastasize from the primary tumor, cancer cells must digest components of the extracellular matrix and basal lamina that normally inhibit migration of cells. Metastasis is thought to be controlled by a large number of genes, including those that encode cell adhesion molecules and proteolytic enzymes.  Metastasis Proteolytic enzymes such as metalloproteinases are present at higher than normal levels in highly malignant tumors and are not susceptible to the normal controls conferred by regulatory molecules such as tissue inhibitors of metalloproteinases (TIMPs).  Predisposition to Some Cancers Can Be Inherited Most cancers result from somatic cell mutations, but 50 forms of hereditary cancer are known.  Cancer susceptibility genes Most inherited cancer-susceptibility genes are not sufficient in themselves to trigger cancer development. At least one other somatic mutation in the other copy of the gene must occur to drive a cell toward tumorigenesis.  FAP Mutations in other genes are also usually necessary to fully express the cancer phenotype. An example is the development of familial adenomatous polyposis (FAP). apc mutation (inherited) ras mutation, lose dcc, mutation, cell finally, loss of p53 partially escape cell cell-to-cell contact adhesion and also cycle control inhibition differentiation Viruses Contribute to Cancer in Both Humans and Animals 15% of cancers are causes by viruses, biggest risk factor next to smoking Most animal viruses that cause cancer are retroviruses. Retroviruses integrate into the host genome as a provirus that is replicated with the host’s DNA during the normal cell cycle.  A proto-oncogene can be converted to an oncogene upon incorporation into a retrovirus in two ways: – the gene sequence may be altered or truncated such that it has abnormal activity – the gene may be brought under the control of powerful promoters and enhancers such that the product is made in excess, or in inappropriate circumstances. Retroviruses can also exert oncogenic effects via insertion into the host genome at sites close to or within proto- oncogenes. This genetic disruption is referred to as insertional mutation. Gardasil Environmental Agents Contribute to Human Cancers Any substance or event that damages DNA has the potential to be carcinogenic if it causes mutations to occur in proto-oncogenes or tumor suppressor genes.  Chemical Carcinogens Some chemical carcinogens act directly on DNA Others become damaging only after they have been changed to a more reactive form by metabolic processes. – Intracellular enzymes, cytochrome P-450 oxidases, normally help convert ingested toxins into harmless secreted products. Their activity on certain compounds (including benzopyrene, present in coal tar and tobacco smoke) generates products that are highly mutagenic. Metabolic activation of a carcinogen. Example: aflatoxin B1, is a toxin from a mold (Aspergillus flavus oryzae). may contribute to liver cancer in the tropics is associated with characteristic mutations of the p53 gene AMES test 55  New therapies are emerging from our knowledge of cancer biology. Antibodies against Her2, an EGF RTK expressed on breast cancer cells. Gleevec Inhibitors of VEGF. Inhibitors of Jak kinases Checkpoint inhibitors Genetically engineered T cells (CAR-T (chimeric antigen receptor T cells) Multidrug treatments may be more effective than sequential treatments.