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Genetic factors and cell transformation CEEPUS 2004 Zadar, Croatia Eliška Marklová Charles University, Faculty of Medicine, Department of Pediatrics Hradec Králové, Czech Republic
C ancer - multi-step and multi-gene disease Cancerogenesis – process – series of events All types of cancers are caused by changes in genes. Cancer - a genetic disease of somatic cells . Cancer - the most common human genetic disease. Not every gene is involved in cancerogenesis! Various independent gene mutations (> 5) - breakdown of several pathways.
Predominant mechanisms for cancers are : t h ese changes misregulation of gene transcription expression of the protein: - with a transforming potential - in an inappropriate place or/and time - appearance of a novel fusion protein decreased apoptosis mutation in a ngiogenic gene s ( cell's blood supply ) mutation in m etastasis genes ( spread of cancer ) impairment of a DNA repair pathway transformation of normal genes into oncogene s malfunction of anti-oncogene s (tumor suppressor genes)
Cellular control depends on a constant balance Imbalance : proto-oncogenes over-expressed , anti-oncogenes inactivated or lost. between proto-oncogenes - favour cell reproduction, and anti-oncogenes - prevent cell reproduction.
Proto-oncogenes - normal cellular regulatory genes that encourage the growth of a cell ( regulate cell division, DNA synthesis & repair , suppress tumour formation ). Various groups proto-oncogenes for: ( 100 g . f ; 13 fam .) growth growth f cellular trenscrip. transcrip. DNA factors receptors kinases factors proteins bind.pp sis, PDGF erB-1,-2; src, abl c-,L-,N - myc K-,H-,N- ras SNF2
Oncogenes - altered versions of proto-oncogenes with potential to cause cell transformation ;
cellular c-onc (in genome of the cancer cell),
viral v-onc (brought by an infecting virus ). “nuclear” + “membrane” oncogene necessary (v-ras + c-myc)
Proto-oncogenes – oncogenes accelerator on a car Ca ncer - dominant mode of expression - 1 mutant allele of g ene .
Many proto-oncogenes encode proteins in the growth factor signalling machinery
“ growth factors” are extracellular proteins signalling cells to divide (differentiate, migrate and die)
there are over a 100 growth factors, over 13 families
specific interaction with high-affinity cellular receptors (which typically span the membrane) with intracellular signalling properties (tyrosine kinase)
occupancy of the receptor triggers an intracellular signalling cascade
gene over-expression ( without sequence alter; P63 – lung cancer )
telomerase reactivation (somatic - germ - cancer cells )
rearrangement ( Bloom's sy – chrm. instability; 15q26.1) rearrangement – within 1 chrm. - normal l abnormal ? translocation - between 2 chrm. - abnormal
Specific changes in particular chrms. - often associated with certain types of cancer.
Proto-oncogenes activation - m echanisms :
Pancreatic carcinomas point mutation H uman pancreatic carcinomas mostly show a loss of part of chr m. 18 . Point-mutations are supposed - in the K-RAS and INK4A ( proto-oncogenes ) , p53 and DPC4 / SMAD4 ( anti-oncogenes ). Main alteration - interplay with the cell cycle . All proteins ( K-RAS, INK4a, p 53 and TGF- β ) regulate directly or indirectly G1 progression , to E2F dep endent S phase entry. DPC - deleted in pancreatic cancer ; SMAD – "Sma -2 „ ( M others A gainst D ecapentaplegic ) in Caenorhabditis elegans + D rosophila mad protein There is a whole family of S MAD proteins , cytoplasmic signaling proteins, all involved in signal transducti on of TGF- β ( t ransforming g rowth f actor β ) related pathways. nerve
Retinoblastoma deletion of children - most common intraocular cancer of childhood - some time associated with small deletions at the Rb locus ( anti-onco gene) of chrm . 13 ( q14 ). The first gene deletion can be either inherited by germline change or acquired . The second deletion is random - both required for neoplastic transformation . Structural abnormalities of the Rb gene with absent protein expression - frequent in all types of human acute leukemia , particularly common in M4, M5 myeloid leukemia and in Philadelphia chrm.-positive leukemia . leukocoria –“white pupil“
Gene translocation reciprocal recombination of segments of 2 diff . chrms. Each oncogenic translocation loss of regulation of gene product activity that controls rate of cell division or cell death . Tumor specific translocations: breakpoint - within a gene enhancing/ destroying its functio n: t( 8;14) Burkitt‘s lymph ; t(14;18) bcl-2 , ( apoptosis), t(15;17) APML (pml/rar ) (chimeric transcript.f . ) breakpoint - create a hybrid gene with new function : t( 9;22) CML bcr/abl, Ph 1 chrm - ( Tyr kinase a ctivity ) t (11;4) MLL APML - acute promyelocytic leukemia; CML - chronic myelocytic leucemia; MLL - m yeloid/lymphoid or mixed-lineage leukemia
Burkitt’s lymphoma translocation + enhancer r are cancer in young children (ce ntral Africa ); a ssociat ion with Epstein - Barr virus infection ? Burkitt’s lymphoma: c-Myc proto-oncogene ( chrm . 8 ) translocation to chrm . 14 (2, 22), close to the enhancer for the heavy (light) chain genes , and is activated . Result: overproduction of the c-Myc product ( transcription factor ) uncontrolled cell division. The „ antibodies genes “– Ig chrm . 14 ( heavy chain), chr m . 2 (kappa light chains) chr m . 22 (lambda light chains) - expressed in B-cells ( transcription factors for the promoters and enhancers needed to turn the genes „ on “)
Fusion of two proteins Chromosomal breaks and fusions lead to novel “chimeric” protein generation new splice combinations; novel protein often looses the regulatory/inhibitory domain of the original protein. R eciprocal translocations two new fusion proteins .
Leukemia, chronic myeloid translocation + fusion The resulting fusion gene BCR/ABL codes for chimeric signal molecule - fusion protein with Tyr kinase activity ; activates signal transduction pathways uncontrolled cell growth. CML - specific abnormality Philadelphia (Ph 1 ) chr m. - translocation between long arms chr m. 9 - proto-oncogene A BL , A b leson l eukemia virus - and chrm. 22 - B CR gene, b reakpoint c luster r egion Many subtypes of T-cell or B-cell leukemias - associated with abnormalities, incl . translocations ,that transpose T CR ( T-cell receptor ) gene or Ig ( Immunoglobuline) gene into proto-oncogene proximity activat ion .
Oncogenes and cell death apoptosis A variety of environmental stimuli can lead to the activation of apoptosis or programmed cell death . C ancers - characterized by rates of apoptosis . DNA damage , growth factor withdrawal + oncogene activation + heat shock + activation of death receptors activation of caspase cascade and cell death. Cancers - n ot cell proliferation - rate of cell dea th. PASG - Proliferation-associated SNF2-like g ene = SNF2 family of proteins = DNA dependent ATPases involved in transcription, recombination, and DNA repair ( activity of other genes by methylation, or modifying histones . SNF - sucrose non-fermenter
B CL -2 in low grade lymphoma translocaion over-expression - apoptosis O ver - expressed is proto-oncogene B CL -2 ( as a result of reciprocal translocation t ( 14;18 ) with the antibody IgG heavy chain locus chr m 14 ) . BCL-2 gene gets close to the heavy chain gene enhancer , very active in B - cells. B CL -2 ( B-cell s leukemia ) = membrane protein ( EPR , nuclear, mitochondrion ) . F unction : anti-apoptotic factor (retards cell deat h – blocks cell suicide lymphoma ). BCL-2 is an inhibitor of the protease cascade that leads to cell death ; it is over - expressed in various cancers .
What makes BCL-2 a proto-oncogene? Genes encoding inhibitors of apoptosis can act as oncogenes . B -cells, like all activated lymphocytes, die a few days after they do their job; aging B-cells kill themselves by apoptosis . However, high levels of the BCL -2 protein protect the cells from early death by apoptosis , preventing the activation of the caspases carry ing out the proces s.
DNA-repair pathway New breast cancer DNA repair gene involved in 4% of all female cancers. In 1994, two breast cancer susceptibility genes were identified: BRCA1 on chr m. 17 BRCA2 on chr m. 13 (normally participating in repairing radiation-induced breaks in DNA ). Mutation - risk of breast or ovarian cancer . Fate of DNA damage : tolerated (ignored), kill the cell /cell itself; fixed mutation; repaired - DNA repair mechanisms ; if failed, than permanently altered genes to offspring .
MLH1 - chrm 3
MSH2 , MSH6 , PMS1 - chrm 2
PMS2 - chrm7
work together as a protein complex to repair DNA damage
MSH2 PMS1 PMS2 MSH6 MLH1
How does a BRCA gene mutation cause cancer susceptibility? cell makes 50% of usual amount of BRCA protein cell functions normally second hit result: no BRCA protein is made DNA not repaired BRCA gene BRCA protein DNA damage repair with time ... inherited BRCA1 gene mutation new BRCA1 gene mutation inherited BRCA1 gene mutation DNA damage cell with no BRCA protein BRCA1 protein cells blood vessel DNA damage dysplasia Cancer cells
How does the loss of BRCA protein cause cancer susceptibility?
DNA repair defective
disruption of p53 function
accumulation of new DNA gene mutations
loss of tumour suppressor genes
activation of oncogenes
proliferation of mutant cell types
hyperplasia dysplasia carcinoma
Colon cancers DNA repair Colon cancer is one of the most common inherited cancer syndromes known. W hy mutations in genes that are essential in all tissues preferentially cause cancer in the colon ? E nvironmental factors play a role in colon cancer incidence ? Among the genes found to be involved in colorectal cancer are: MSH2 and MSH6 on chr m . 2 and MLH1 on chr m . 3 . Normal function of their protein products: to repair mistakes made in DNA replication . Mutation the replication mistakes are not repaired damaged DNA colon cancer. MSH - MLH - mutL homolog 1, colon cancer
Telomerase reactivation Telomeres , the ends of chrms - special repeat sequenc e - decrease in length with each somatic cell division (a "mitotic clock" - counting the total number of divisions; critical minimum signals the cell to stop the division cycle); ensure chrm. s tability ; telomerase . In contrast, germinal cells - immortalized cells - capacity to proliferate indefinitely, and human cancer cells do not show shortening of telomere or a loss of telomerase activity. Telomerase is reactivated in immortal cells + cancer cells . Association with TGF- (t ransforming growth factor- β ) and BCL-2 (B-cells leukemia) genes expression in breast cancer ? Normal somatic cells - programmed for a limited number of cell divisions; telomerase a ctivity slowly decreases.
A nti - oncogenes - tumor suppressor genes brake on a car (RB p53 DCC APC MCC NF-1 WT-1) negative regulators of cell division - in contrast to oncogenes. Cancer - inactivation of genes ( inhibit . cell division ).
M echanisms :
genetic (chromosome or microsatellite instability - point mutation, deletion ) - checkpoint inhibitor such as p53 , or a transcription inhibitor , such as Rb ( retinoblas toma assoc.)
epigenetic (hyper methylated promoters , CDKN2A , p16) . P romoter methylation represents significant contributor to human carcinogenesis ; 9 % of Rb1 in retinobl ; 84 % of MLH1 colorectal ca
For most cancers - the more frequently mutated genes are the anti - oncogenes (* exception leukemias , lymphomas ). Recessive mode of expression - 2 alleles of the gene altered . CDKN - c yclin-depend . kinase inhibitor 2A ( p16, inhibits CDK4)
• replication of damaged DNA • increased frequency of mutation
loss of p53 mediated apoptosis
loss of p16 in a variety o f tumours over expression of cyclinD can uncouple cell cycle control Bcl1 / PRAD1 oncogene translocated cyclinD ( next to Ig promoter in B cell lymphoma ) DNA DAMAGE
Lung carcinoma, small cell oncogenes + anti- Lung cancer is the most common cause of cancer deaths (men / women). Small cell lung carcinoma is distinctive from other kinds of lung cancer (metastases present at the time of discovery). As with other cancers, mutations in oncogenes ( deletion of part of chr m. 3 ) and tumor - suppressor genes ( inactivation of Rb gene = inhibitor of cell cycle G1) that control cell growth and division - are observed .
Neurofibromatosis oncogene + anti-oncogene Type 2, ( m utation and/or loss of NF2 - tumour-suppressor gene , normally a brake on cell growth and division, ensuring that cells do not divide uncontrollably) . occurs sporadically or on a familial basis with autosomal dominant inheritance ; 8 classified subtypes - the two most distinct forms: type 1 ( chrm . 17 ) and type 2 ( chrm . 22 ): acoustic or central; 8 th nerve tumors; schwannomas and multiple meningiomas ; often + other intracranial or intraspinal tumors.
Malignant melanoma anti- oncogenes Malignant melanoma is the most aggressive kind of skin cancer ( sudden change in skin growth ; appearance of a mole ).
study of p16
M utation in the CDKN2 gene on chr m. 9 p21 can underlie susceptibility to melanoma. CDKN2 codes for a protein called p16 ( an important regulator of the cell division cycle - G1 ; it stops the cell from synthesizing DNA before it divides ) . If p16 is not working properly (hypermethylation?), the skin cell does not have this brake and go on to proliferate unchecked. CDKN2 - Cyclin-dependent kinase 4 inhibitor 2A (inhibits CDK4)
Proto-oncogenes, O ncogenes, A nti-oncogenes Mutations - results Point mutation – Lung , Pancreatic , O varian cancer Deletion – Retinoblastoma Translocation + enhancer - Burkitt lymphoma Translocation + fusion – Leukemia , CML Over-expression – BCL-2, lymphoma Amplification - Telomerase reactivation - BCL- 2 , Breast cancer ? DNA repair defect – BRCA, Colon cancer Apoptosis inhibition - BCL -2 Hypermethylation - Malignant melanoma Neuroblastoma; Neurofibromatosis