(优选)第二十章癌基因和抑癌基因课件

1、（优选）第二十章癌基因和抑癌基因Measuring transformation in-vitroNormaltransformed 肿瘤是机体在各种致瘤因素的作用下，局部组织的细胞在基因水平上失掉了对其生长的正常调控，导致异常增生而形成的新生物。 致瘤因素 正常细胞 基因改变 肿瘤细胞Development of CancerDefective cell cycles checkpoints mechanisms allows errors in the cell duplication process to persist into the next generation and can

2、lead to and regulated proliferation and the development of cancer.Two different types of mutations contribute to cancer formation: activating mutations in proto-oncogene and inactivating mutations in tumor suppressor genes.OncogenesStimulate ProliferationInhibit DifferentiationInhibit ApoptosisTumor

3、 Suppressor GenesInhibit ProliferationPromote DifferentiationStimulate Apoptosis一、癌基因的基本概念癌基因（oncogene）是指存在于正常细胞内，与细胞生长发育调控有关的一组结构基因。癌基因发生结构异常或表达异常时，可以引起细胞癌变。癌基因可按其来源不同而分为病毒癌基因（v-onc）和细胞癌基因（c-onc）。 Oncogenes were first discovered in certain retroviruses and were later identified as cancer-causing ag

4、ents in many animals First link between viruses and cancer proposed by Francis Peyton Rous in 1910 (Nobel Prize, 1966): cell-free extracts from chicken tumors injected into healthy chickens caused new tumors.History of oncogeneRous Peyton ：The 1966 Nobel Prize in Physiology or Medicine鸡肉瘤病毒基因组结构图 调节

5、和启动转录 LTR gag pol env src LTR 长末端重复序列癌基因正常的病毒基因产生病毒核心蛋白产生逆转录酶和整合酶 产生病毒 外膜蛋白产生酪氨酸蛋白激酶Rous Sarcoma Virus (RSV)Discovered by Harold Varmus and Bishop, 1975-76 (Nobel Prize, 1989).A transforming retrovirus: a cancer-causing single-stranded RNA virus that uses reverse transcriptase enzyme to make ssDNA,

6、then ds DNA, which integrates into host DNA.Note: not all oncogenes caused by viruses.100s of oncogenes now known.Human T-cell leukemia virus (HTLV) is a human RV; codes a TF.Retroviral life cycleFigure 6 Retroviral life cycleRetroviruses have two identical copies of a plus single-stranded RNA genom

7、e and an outer envelope containing protruding viral glycoproteins. After envelope glycoproteins on a virion interact with a specific host-cell membraneprotein or group of proteins, the retroviral envelope fuses directly with the plasma membrane without first undergoing endocytosis (step 1). Followin

8、g fusion, the nucleocapsid enters the cytoplasm of the cell; then deoxynucleoside triphosphates from the cytosol enter the nucleocapsid, where viral reverse transcriptase and other proteins copy the ssRNA genome of the virus into a dsDNA copy (step 2). The viral DNA copy is transported into the nucl

9、eus (only one host-cell chromosome is depicted) and integrated into one of many possible sites in the host-cell chromosomal DNA (step 3). The integrated viral DNA, referred to as a provirus, is transcribed by the host-cell RNA polymerase, generating mRNAs (light red) and genomic RNA molecules (dark

10、red). The host-cell machinery translates the viral mRNAs into glycoproteins and nucleocapsid proteins (step 4). The latter assemble with genomic RNA to form progeny nucleocapsids, which interact with the membrane-bound viral glycoproteins,. Eventually the host-cell membrane buds out and progeny viri

11、ons are pinched off (step 5). Origin of Transforming Retroviruses病毒癌基因v-src来源于宿主细胞的C-SRC基因 逆转录复制整合转录感染病毒RNARNA-DNA前病毒DNA细胞基因组 DNA病毒RNA-癌基因RNA病毒颗粒宿主细胞再感染宿主细胞携带癌基因的病毒颗粒RNA病毒与宿主细胞基因组整合过程示意图Proto-oncogenes癌变因此，病毒癌基因（v-onc）就是指一类存在于肿瘤病毒中，能使宿主细胞发生恶性转化的基因。 病毒癌基因的特点 病毒癌基因无内含子,而原癌基因通常有内含子或插入序列。病毒癌基因较原癌基因有较强的转

12、化细胞功能，病毒 癌基因与同源的原癌基因在外显子序列中存在着微小的差别。病毒癌基因常会出现碱基取代或碱基缺失等突变,而原癌基因则较少发现这类突变。病毒癌基因通常丢失了原癌基因两端的某些调控序 列，而在病毒高效启动子作用下有较高的转录活性。二、细胞癌基因 细胞癌基因(c-onc)又称为原癌基因（proto-oncogene），存在于细胞基因组中、正常情况下处于静止或低水平（限制性）表达状态，对维持细胞正常功能具有重要作用，当受到致癌因素作用被活化而导致细胞恶变的基因。细胞癌基因的特点：广泛存在于生物界基因序列高度保守表达产物对细胞正常生长、繁殖、发育和分化起着精确的调控作用。基因结构发生异常或表

13、达失控，导致细胞生长增殖和分化异常二、癌基因活化的4种机制示意图 指来源于病毒等的启动子或增强子插入到细胞癌基因的附近或内部而使其开放并异常转录。如鸡白细胞增生病毒引起的淋巴瘤，就是由于病毒的DNA序列整合到宿主细胞c-myc基因附近，成为该基因的强启动子，导致c-myc基因过强表达。（一）插入激活c-myc原癌基因的插入激活 即基因数量或拷贝数目明显增加。常见的为myc原癌基因，由于其基因数目的增多而使其表达的蛋白产物增多。（二）基因扩增 常见的为ras原癌基因的点突变。 ras原癌基因点突变后导致其GTPase活性下降，不能将其结合的GTP迅速水解，从而使其持续保持激活状态。 正常细胞 H

14、-ras 基因碱基序列 ATG ACG GAA TAT AAG CTG GTG GTG GTG GGC GCC GGC GGT GTG肿瘤细胞 H-ras 基因碱基序列 ATG ACG GAA TAT AAG CTG GTG GTG GTG GGC GCC GTC GGT GTG正常细胞 P21 蛋白氨基酸序列 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Ala Val肿瘤细胞 P21 蛋白氨基酸序列 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Ala ValH-ras基因的点突变 （三）点

15、突变Ras Proto-oncogeneMutated in 30% of all cancers.A “molecular switch” in the signal transduction pathway leading from growth factors to gene expression controlling cell proliferation: GF receptor Ras TF target genes growth.A single amino acid change in Ras protein can cause constant stimulation of

16、the pathway, even in the absence of growth factors.由于染色体重排而导致细胞癌基因从正常位置转移到另一位置，常常是插入一启动子后而使其转录活性增加。Burkitt淋巴瘤中，含有c-myc的8号染色体易位，与14号染色体连接，靠近免疫球蛋白肽链的基因，与该区活性很高的启动子连接而受到活化。c-abl原癌基因，经重排后插入到另一称为bcr基因的启动子之后，而使其转录活性增加，从而引起慢性粒细胞性白血病的发生。 （四）染色体易位Chromosomal Translocation that creates Philadelphia Chromosome

17、BCR-ABL Oncogene: Breaks in ABL Gene of Chromosome 9 andBCR Gene of Chromosome 22Fusion Protein causes Chronic Myelogenous Leukemia出现新的表达产物 出现过量的正常表达产物出现异常、截短的表达产物 癌基因激活的结果：不同癌基因有不同的激活方式，一种癌基因也可有几种激活方式： c-myc、ras肿瘤细胞中常发现两种或多种细胞癌基因的活化，如白血病细胞株HL-60中有c-myc和N-ras的同时活化。例：原代培养大鼠胚胎成纤维细胞作用于细胞膜上的受体系统或直接被传递至细

18、胞内，通过蛋白激酶活化转录因子，引发一系列基因的转录激活。三、原癌基因的产物与功能sis表达蛋白P28和PDGF一样能促进血管的生长。（一）细胞外生长因子 例如：（二）跨膜的生长因子受体：接受细胞外的生长信号并将其传入胞内。受体的胞质结构区具有特异的蛋白激酶活性，通过磷酸化作用使其结构发生改变，增加激酶对底物的活性，促进生长信号在胞内的传递。 例如 EGFR、HER2有酪氨酸特异的蛋白激酶活性。非受体酪氨酸激酶(src，abl)、丝氨酸/苏氨酸激酶(raf)，ras蛋白(H-ras，K-ras和N-ras)及磷脂酶(crk产物)。（三）细胞内信号传导分子原癌基因的产物作为胞内信息传递体系成员，

19、或者通过影响第二信使作用，将接受到的信号由胞内传至核内，促进细胞生长。 例如：（四）核内转录因子某些癌基因表达蛋白定位于细胞核内，与靶基因的顺式调控元件相结合直接调节靶基因的转录活性。 例如：c-fos是一种即刻早期反应基因(immediate early gene，IEG)。作为传递信息的第三信使。 生长因子 生长因子受体 蛋白激酶及其他信号转导组分 细胞周期蛋白 细胞凋亡调控因子 转录因子原癌基因BRAF所编码的蛋白质属于丝/苏氨酸激酶，是MAPK信号通路的重要组成分子，在调控细胞增殖、分化等方面发挥重要作用。四、癌基因表达产物促进肿瘤发生发展约60%的黑素瘤中BRAF发生突变，其第600

20、位氨基酸从缬氨酸突变为谷氨酸（V600E）最为常见，导致B-Raf的持续激活。（一）BRAF 例如：分子靶向药物 威罗菲尼VemurafenibHER2是表皮生长因子受体家族成员，具有蛋白酪氨酸激酶活性，能激活下游信号通路，从而促进细胞增殖和抑制细胞凋亡。在30%的乳腺癌中HER2基因发生扩增或者过度表达，其表达水平与治疗后复发率和不良预后显著相关。（二）HER2例如：单克隆抗体药物 赫塞汀Herceptin慢性粒细胞白血病患者的9号染色体与22号染色体之间发生易位，从而融合产生了癌基因BCR-ABL，编码的蛋白质Bcr-Abl具有持续活化的蛋白酪氨酸激酶活性，能促进细胞增殖，并增加基因组的不

21、稳定性。在95%的慢性粒细胞白血病患者中都伴随有BCR-ABL融合基因的产生，在一些急性淋巴白血病患者中也有发现。（三）BCR-ABL 例如：分子靶向药物 伊马替尼 ImatinibAcquired mutations of oncogenes Most cancer causing mutations involving oncogenes are acquired, not inherited. They generally activate oncogenes by chromosome rearrangements, gene duplication, or mutation. For

22、 example, a chromosome rearrangement leads to formation of the gene called BCR-ABL. This leads to the disease chronic myeloid leukemia (CML). the gain-of-function mutations that convert proto-oncogenes to oncogenes act dominantly; that is, mutation in only one of the two alleles is sufficient for in

23、duction of cancer.Inherited mutations of oncogenes A few cancer syndromes are caused by inherited mutations of proto-oncogenes Multiple endocrine neoplasia type 2 (多发性内分泌瘤) is caused by an inherited mutation in the gene called RET. Inherited mutations in the gene called KIT cause hereditary gastroin

24、testinal stromal tumors (胃肠道间质瘤, GIST). Inherited mutations in the gene called MET cause hereditary papillary renal cancer（乳头状肾癌）. Cancers Usually Result from a Series of Mutations in a Single CellColon Cancer:MSH2和MLH1基因失活染色体：改 变： 基 因： 5q 突变或缺失 FAP、APC、MCC？12q 突变 K-ras18q 缺失 DCC17q 缺失和突变 p53正 常肠上皮高

25、增殖肠上皮早期腺瘤中期 腺瘤晚期 腺瘤腺癌转 移 癌nm23突变DNA低甲基化其他基因的改变（如TGF-受体）Tumor Suppressor Genes Tumor Suppressor genes: are genes that act to inhibit cell proliferation and tumor development. If Tumor Suppressor Gene is Mutated Inactivated It will lead to cell transformation 肿瘤抑制基因(tumor suppressor gene) 肿瘤抑制基因的概念也称抗

26、癌基因（anticancer gene）或抑癌基因，是调节细胞正常生长和增殖的基因。当这些基因不能表达，或者当它们的产物失去活性时，细胞就会异常生长和增殖，最终导致细胞癌变。反之，若导入或激活它则可抑制细胞的恶性表型。Discovery of Tumor SuppressorsFirst discovered in 1960s by Henry Harris. Harris fused tumor cells with normal cells and discovered some of the hybrid cells were normal. Harris hypothesized th

27、at the normal cells contained gene products that suppressed uncontrolled cell proliferation.Five broad classes of proteins encoded by tumor-suppressor genesIntracellular proteins, such as the p16 cyclin-kinase inhibitor, that regulate or inhibit progression through a specific stage of the cell cycle

28、.Receptors for secreted hormones (e.g., tumor derived growth factor ) that function to inhibit cell proliferation.Checkpoint-control proteins that arrest the cell cycle if DNA is damaged or chromosomes are abnormal.Proteins that promote apoptosis.Enzymes that participate in DNA repair.Familial cance

29、r - RbSporadic retinoblastoma 60% of retinoblastoma cases. Develops in children with no family history. Occurs in one eye. Hereditary retinoblastoma 40% of retinoblastoma cases. Onset typically is earlier than sporadic cases. Multiple tumors involving both eyes.Retinoblastoma (Rb) caused bymutated R

30、b geneThe First Tumor-Suppressor Gene 视网膜母细胞瘤Alfred Knudsons 2-hit modelTwo mutations are required for the development of retinoblastoma. Sporadic retinoblastoma Child starts with two wild type alleles (RB+/RB+). Both alleles must mutate to produce the disease (RB/RB). Probability of both mutations

31、occurring in the same cell is low; only one tumor forms (e.g., one eye). Hereditary retinoblastoma Child starts with heterozygous alleles (RB/RB+). Only one mutation is required to produce disease (RB/RB). Mutations resulting in loss of heterozygosity (LOH) are more probable in rapidly dividing cell

32、s, and multiple tumors occur (e.g., both eyes).Loss of heterozygosity (LOH)杂合性缺失Rb基因的表达产物为P105Rb，在细胞内存在低磷酸化型（活性型）和高磷酸化型（非活性型）两种类型。不同类型的P105Rb对转录因子E2F有不同的亲和力。Rb 基因的作用机制G0 G1期Rb蛋白E-2FS期E-2FDNAmRNADNAPRb蛋白Tumor Suppressors are Recessive Tumor suppressors must be inactivated This means both copies must

33、be lost/mutatedTumor suppressors and familial cancerHereditary cancer is caused by the inheritance ofone copy of a defective tumor suppressor1. Antagonize the action of oncogenes eg. p53 is activated by oncogenes. p53 protects against cancer by inducing cell cycle arrest and/or apoptosisFunctions of

34、 Tumor Suppressor genesmyccell growthp532. Block proliferation: Cell cycle inhibitors: eg. Rb blocks entry into S phase by binding to and inhibiting RB. INK-4 gene: that produces P16 that inhibits cdk4/cyclin D action ( to phosphorylate Rb gene to inactivate its action) Repressor transcription facto

35、rs: e.g.; WT1 is a repressor that appears to suppress transcription factor ( Insulin like growth factor) which will contribute in the development of tumor. Activator transcription factors: e.g.; SMAD family that are activated by TGF-, leading to inhibition of cell proliferation . P53: that produces

36、P21 that has the same action of P16 in inhibiting the action of cdk/cyclin. Functions of Tumor Suppressor genesPTEN通过阻断PI3K/AKT信号通路抑制细胞的生长 3. Induce apoptosis: Form of cell suicide. A cell which has lost growth control will often undergo apoptosis. Cell damage or stress can also lead to apoptosis. p

37、53 is a critical regulator of apoptosis. Transcription factor which activates pro-apoptotic moleculesFunctions of Tumor Suppressor genes Most commonly mutated gene in cancers (50% of total). When p53 is mutated, DNA-damaged cells are not arrested in G1 and DNA repair does not take place. This failur

38、e to arrest DNA-damaged cells will be repeated in subsequent cell cycles permitting other mutations to accumulate, culminating in neoplastic transformation. tumor formation and cancer.p53 Mutationsp53的结构及其在清除DNA损伤细胞中的作用 p53 the guardian ofthe genomeRegulation of the cell cycle4. DNA Repair DNA repai

39、r prevents the accumulation of mutations Defects in DNA repair genes leads to genomic instability Accelerates the activation of oncogenes and the loss of tumor suppressors Many cancer prone syndromes associated with defects in DNA repair, BRCA1, ATM, MRE11, NBS,Functions of Tumor Suppressor genes A

40、small proportion of breast cancer is heritable. Two genes are associated with predisposition to breast cancer. BRCA1 on chromosome 17 BRCA2 on chromosome 13 Normal function of both is in repair of ds DNA breaks.Breast Cancer Tumor SuppressorsTumor suppressor genes- summaryTumor suppressor genes inhi

41、bit oncogenes suppress proliferation Induce cell death repair DNA prevent mutation These are “loss of function” or recessive mutations. Responsible for hereditary forms of cancer Being heterozygous enhances the probability of cancer but this will require a mutation in the corresponding other allele.

42、 e.g., it need to be homozygous for the gene.Tumor suppressor genes- summaryp53 A transcription factor that regulates genes controlling cell division and cell death. Important in the cellular response to DNA damage. Aids in decision between repair and induction of cell death.Rb Functions by altering

43、 transcription factor activity. Contributes to the control of cellular division by acting as an inhibitor.APC The APC protein binds and stimulates the degradation of a transcription factor. Absence of functional APC protein leads to increased cell division.BRCA BRCA proteins have multiple functions

44、including repairing DNA damage and regulation of gene expression. Non-functional BRCA leads to compromised DNA repair and gene regulation.促进正常细胞向肿瘤细胞转化的因素 生长因子Growth factors 第三节 生长因子(growth factor)一类由细胞分泌的、类似于激素的信号分子，多数为肽类（含蛋白类）物质，具有调节细胞生长与分化的作用。内分泌 (endocrine)旁分泌 (paracrine)自分泌 (autocrine) 作用模式 ： 生

45、长因子分泌后，通过血液运输作用于远端靶细胞细胞分泌生长因子作用于邻近其它类型细胞，对自身细胞不发生作用生长因子作用于合成及分泌该生长因子的细胞本身一、生长因子的分类和功能（一）生长因子的分类生长因子名称组织来源功能表皮生长因子（EGF）唾液腺、巨噬细胞、血小板等促进表皮与上皮细胞的生长，尤其是消化道上皮细胞的增殖肝细胞生长因子（HGF）间质细胞促进细胞分化和细胞迁移促红细胞生成素（EPO）肾调节成红细胞的发育类胰岛素生长因子（IGF）血清促进硫酸盐参入到软骨组织促进软骨细胞的分裂、对多种组织细胞起胰岛素样作用神经生长因子（NGF）颌下腺含量高营养交感和某些感觉神经元、防止神经元退化血小板源生长

46、因子（PDGF）血小板、平滑肌细胞促进间质及胶质细胞的生长、促进血管生成转化生长因子（TGF-）肿瘤细胞、巨噬细胞、神经细胞作用类似于EGF，促进细胞恶性转化转化生长因子（TGF-）肾、血小板对某些细胞起促进和抑制双向作用血管内皮生长因子（VEGF）低氧应激细胞促进血管内皮增殖和新生血管分化（二）生长因子的功能生长因子的生物学效应主要表现在促进细胞生长、分化、促进个体发育等方面。但是有些生长因子具有双重调节作用或负调节作用。 同一生长因子对不同细胞的作用有所不同 一种细胞也可受不同生长因子调节 具有负调节作用的生长因子比较少，人们通常把这种负调节因子（negative growth facto

47、r）称为细胞生长抑制因子。 产生相应第二信使胞内相关蛋白质被磷酸化与膜受体结合酪氨酸激酶活化蛋白激酶活化 核内转录因子活化基因转录与胞内受体结合生长因子 - 受体复合物，活化相关基因二、生长因子作用机制The seven types of proteins that participate in controlling cell growthgrowth factors (I) growth factor receptors (II) signal-transduction proteins (III)transcription factors (IV) pro- or anti-apoptotic proteins (V)cell cycle control proteins (VI)DNA repair proteins (VII)Class VII mutations greatly increase t