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Download Introduction Lecture The Biology of Cancer
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Transcript
The Biology of Cancer • The cells of multicellular organisms are committed to the survival of the germ cells • Cells collaborate for germ cell survival • Mutations giving rise to “selfish behavior” jeopardizes the survival of the germ cells • Cancer is that selfish cell Cancer • Reproduce in defiance of the normal restraints (proliferation) • proliferation gives rise to a tumor (neoplasm), a growing mass of abnormal cells • If the cells remain clustered in a single mass, it is benign and can be removed with surgery • Invade and colonize territories normally reserved for other cells • A tumor is counted as cancer only if it is malignant, the ability to invade surrounding tissue • The formation of secondary tumors is called metastases Classifications of Cancer • Cancers are classified according to the tissue and cell type from which they arise • Cancers arise from: – Epithelial cells: carcinoma – Connective tissue/muscle: sarcoma – Hemopoietic cells: leukemias Incidence of Cancers Estimated New Cases Both Sexes All Sites Oral cavity & pharynx Male Estimated Deaths Female 1,372,910 710,040 662,870 Both sexes Male Female 570,280 295,280 275,000 29,370 19,100 10,270 7,320 4,910 2,410 Tongue 7,660 5,050 2,610 1,730 1,120 610 Mouth 10,070 5,370 4,700 1,890 1,100 790 Pharynx 8,590 6,520 2,070 2,130 1,490 640 Other oral cavity 3,050 2,160 890 1,570 1,200 370 253,500 134,370 119,130 136,060 75,020 61,040 Digestive system Esophagus 14,520 11,220 3,300 13,570 10,530 3,040 Stomach 21,860 13,510 8,350 11,550 6,770 4,780 5,420 2,840 2,580 1,070 580 490 Colon 104,950 48,290 56,660 28,540 27,750 Rectum 40,340 23,530 16,810 Anus, anal canal, & anorectum 3,990 1,750 2,240 620 230 390 Liver & intrahepatic bile duct 17,550 12,130 5,420 15,420 10,330 5,090 Gallbladder & other biliary 7,480 3,330 4,150 3,340 1,270 2,070 32,180 16,100 16,080 31,800 15,820 15,980 5,210 1,670 3,540 2,400 950 1,450 Small intestine Pancreas Other digestive organs 56,290 Incidence of Cancers Estimated New Cases Both Sexes Respiratory system Larynx Male 184,800 102,420 Estimated Deaths Female Both sexes Male Female 82,380 168,140 93,990 74,150 9,880 7,920 172,570 93,010 2,350 1,490 860 860 540 320 Bones & joints 2,570 1,480 1,090 1,210 670 540 Soft tissue (including heart) 9,420 5,530 3,890 3,490 1,910 1,580 66,000 37,580 28,420 10,590 6,920 3,670 59,580 33,580 26,000 7,770 4,910 2,860 6,420 4,000 2,420 2,820 2,010 810 1,690 211,240 40,870 Lung & bronchus Other respiratory organs Skin (excluding basal & squamous) Melanoma-skin Other nonepithelial skin Breast 212,930 1,960 3,770 2,960 810 79,560 163,510 90,490 73,020 460 40,410 Incidence of Cancers Estimated New Cases Both Sexes Genital system Male Estimated Deaths Female Both sexes Male Female 321,050 241,570 79,480 59,920 31,010 28,910 Uterine cervix 10,370 10,370 3,710 3,710 Uterine corpus 40,880 40,880 7,310 7,310 Ovary 22,220 22,220 16,210 16,210 Vulva 3,870 3,870 870 870 Vagina & other genital, female 2,140 2,140 810 810 Prostate 232,090 232,090 30,350 30,350 Testis 8,010 8,010 390 390 Penis & other genital, male 1,470 1,470 270 270 Urinary system 101,880 71,090 30,790 26,590 17,420 9,170 Urinary bladder 63,210 47,010 16,200 13,180 8,970 4,210 Kidney & renal pelvis 36,160 22,490 13,670 12,660 8,020 4,640 430 320 Ureter & other urinary organs 2,510 1,590 920 750 Incidence of Cancers Estimated New Cases Both Sexes Eye & orbit Male Estimated Deaths Female Both sexes Male 2,120 1,090 1,030 230 Brain & other nervous system 18,500 10,620 7,880 12,760 7,280 5,480 Endocrine system 27,650 7,550 20,100 2,370 1,080 1,290 25,690 6,500 19,190 1,490 630 860 1,960 1,050 910 880 450 430 63,740 33,050 30,690 20,610 Thyroid Other endocrine Lymphoma 110 Female 120 10,930 9,680 Cancer and Genetics • If a single abnormal cell is to give rise to a tumor, it must be able to pass on its abnormality to its progeny • These heritable traits are passed on by DNA Causes of Cancer • Carcinogenesis: generation of cancer causing… • Mutagenesis: changes in DNA sequence • Types of carcinogens: – Chemical: local changes in nucleotide sequence – Radiation: causes breaks in chromosomes – Viruses: introduce foreign DNA Mutations of DNA • In a lifetime, each gene (<25,000) undergoes about 1010 individual mutation events. So why does cancer occur so infrequently? Mutations of DNA • In a lifetime, each gene (<25,000) undergoes about 1010 individual mutation events. So why does cancer occur so infrequently? • Almost all of the mutations are removed or corrected • 1 mutation is not enough. Several must accumulate in the genome with age Growth of Cancer • Cancerous growth often depends on deranged control of cell differentiation or cell death • Result is cells dividing out of control Metastases • Cancer cells loosen their adhesion and escape the tissue of their origin • This involves: – Breaking away from tissue of origin – Burrowing through tissue to a blood supply or lymphatic vessel – Entering circulation – Leaving circulation – Possessing the ability to live in a new environment Drugs • Most chemotherapeutic agents impair mitosis and thus target rapidly dividing cells, e.g. cancer, hair, intestinal lining • Disadvantage to chemotherapy: – Those cells that survive are resistant to that drug and have been artificially selected for further growth Molecular Genetics of Cancer • Cancers are not genetically identical, even within a single tumor! • All cancers involve the disruption of normal restraints on cell proliferation often occurring in a small subset of genes Molecular Genetics of Cancer • Proliferation can be regulated by “start” site of cell division or by apoptosis – Make a stimulatory gene hyperactive • Oncogene: dominant effect (1 allele needed) • Proto oncogene: normal alleles – Oncogenes are identified by placing them into normal cells (transformation) and seeing if the normal cells become tumor cells – Make an inhibitory gene inactive • Recessive effect (requires both alleles) • Both gene copies must be inactivated or deleted • The lost gene is a tumor suppressor gene – Tumor suppressor genes are harder to identify Retroviruses • Example is the Rous sarcoma virus (chickens) – Viral RNA is copied to DNA by the host’s cell machinery and inserted into the host cell’s genome. Can then be inherited by subsequent generations Retroviruses • Proto oncogenes can be converted into an oncogene upon incorporation into a retrovirus – Gene sequence altered so it codes for a protein with abnormal activity – Gene brought under control of powerful promoters/enhancers in the viral genome that causes its product to be made in excess or during inappropriate circumstances Identification of Oncogenes in vitro • Transform normal fibroblast cells with DNA from human tumors – Identified the same oncogenes as identified from retroviral transformations – 25% of all cancers result from mutated members of the ras gene family Proto Oncogenes can become Oncogenes • Deletion or point mutation in coding sequence – Hyperactive protein made in normal amounts • Gene amplification – Normal protein greatly overexpressed • Chromosome rearrangement – Nearby strong enhancer/promoter causes normal protein to be overproduced Oncogene Collaboration • A single oncogene is not usually sufficient to turn a normal cell into a cancer cell • Oncogene collaboration – The synergistic action of 2 or more specific oncogenes are required to make a cell cancerous • E.g. transform mouse egg with ras and/or myc oncogenes Tumor Suppressor Genes • Tumor suppressor genes protect cells • Loss of tumor suppressor genes leads to cancerous condition • Problem: It is harder to find something that is missing! Tumor Suppressor Genes: Rb • Retinoblastoma (Rb): cancer of the retina – Deletion of a region of chromosome 13 – Cancerous cells have defect in both alleles – Other somatic cells are deficient in one allele – Rb gene is missing also in cancers of the lung, breast and bladder – Loss of the Rb gene is a major step in the progression toward malignancy Tumor Suppressor Genes: Rb • Rb is found in all cells and acts as a “brake” on the cell division cycle • Regulation is by phosphorylation • Rb protein (pRb) alternates between the phosphorylated and unphosphorylated states in every cell cycle, being unphosphorylated when not cycling Tumor Suppressor Genes: Rb • When unphosphorylated, pRb is bound strongly to gene regulatory proteins preventing them to promote DNA replication in the nucleus • When pRb is removed, DNA replication and cell division goes unchecked DNA Tumor Viruses • DNA tumor viruses activate the cell’s DNA replication machinery as part of their survival • Viral DNA fails to replicate and becomes incorporated into the host’s genome • If the viral gene that activates the host’s machinery for DNA replication is transcribed, this gene can act as an oncogene DNA Tumor Viruses • Large T antigen from the SV-40 genome – binds protein products of two key tumor suppressor genes of the host cells – These genes are disabled permitting the cell to replicate its DNA and divide uncontrolled • One of these proteins is pRb • The other is p53 p53 Mutations • p53 binds to DNA and induces the transcription of a 21 kD protein (p21) • p21 binds to G1 cyclin and CdK2 proteins, which serve to drive the cell past the G1 checkpoint in the cell cycle, and blocks them – Cell is prevented from progressing into S phase and DNA replication p53 Mutations • When DNA damage occurs, there is an increase in p53 expression: – Delays the cell in G1 so repairs can be made – Cell may enter apoptosis and die • If the cell lacks p53, damaged DNA continues to replicate and is transmitted to the daughter cells • Mutations of the p53 gene are found in 50% of all cancers Colorectal Cancer • Arise from the epithelium lining the colon and rectum • Begins with a benign tumor (adenoma) of the gut epithelium forming a protruding mass of tissue (polyp) • Progression of the disease is very slow (10-35 years) – 75% of colorectal cancers have inactivating mutations in the p53 tumor suppressor gene – 50% have a point mutation in a ras proto oncogene Order of Events Leading to Colorectal Cancer • 1) Inactivation of APC (adenomatosis polyposis coli), a tumor suppressor gene – Detected in small polyps – Increase rate of cell proliferation • 2) ras activation – Rare in small polyps, common in larger ones • 3) DCC (Deleted in Colorectal Carcinoma) & p53 mutations – Common in malignant tumors – Loss of p53 allows the cells to divide uncontrollably and accumulate still further mutations Methods: Immunohistochemistry & ELISA Direct Indirect Effector Function of Antibodies: Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) From Abbas et. al. Cell.& Mol. Imm. 1999, Fig. 14-4 The assay is based on measurement of activity of lactate dehydrogenase (LDH) which is a stable enzyme normally found in the cytosol of all cells but rapidly releases into the supernatant upon damage of plasma membrane. The assay is fast and sensitive. Results can be analyzed by spectrophotometry at 500 nm ADCC Results mAb CHO 31.1 mAb UPC-10 (control) PBMC 50:1 (Peripheral Blood MonoCytes) 70 Percent Cytotoxicity 60 50 40 30 20 10 0 0.1 0.01 0.001 1 10 -4 Concentration (ug/m l) Complement-Dependent Cell-Mediated Cytotoxicity (CDC) In vivo ADCC: Efficacy Study Tumors established with 1x106 cells Dose = 400 ug/injection PBMC = 2x107 cells Results of Animal Studies 3500 CH O31.1 + PBM C hIg + PBM C Mean Tumor Volume (mm3) 3000 CH O31.1 hIg 2500 2000 1500 1000 500 0 0 3 7 10 Day 14 17 21 24 MAb CHO31.1 Inhibits Growth of ASPC-1 Tumors in vivo mAb CHO31.1 Binding Affinity: Cell Based ELISA 0.75x105 cells/well Goat Anti-Human IgG (H+L) AP Conjugate secondary mAb ELISA was evaluated using GraphPad Prism Experiments were carried out in duplicate with values indicated as means; bars, SE. Mean OD 405 nm Tumor Cell Lines: SWS 900 & LS174T mAb: CHO31.1 & Human IgG1 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.01 SW900 + Human IgG1 SWS900 + CHO31.1 LS174T + Human IgG1 LS174T + CHO31.1 Bmax=0.98 Kd=0.44 nM 0.1 1 10 mAb (nM) Red line indicates nonlinear regression curve 100 1000 Flow cytometry Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid.