癌基因、抑癌基因与生长因子

1、第 十 六 章 癌基因、抑癌基因、生长因子（ Oncogene,Tumor Suppressor Gene , and Growth Factors ）安徽医科大学生物化学教研室 汪渊、朱华庆 本 章 提 要 细胞癌基因，抑癌基因、生长因子的基本概念 癌基因活化机制 抑癌基因失活机理 细胞的正常生长与增殖由两大类基因调控：正调节信号 促进细胞生长和增殖，并阻止其发生终末分化。负调控信号 抑制增殖, 促进分化、成熟和衰老，最后凋亡。这两类基因中任何一种或它们共同的变化，即有可能引起细胞增殖失控导致肿瘤的发生。癌基因与抑癌基因的作用机制涉及基因表达调控及细胞分裂、分化。癌基因可编码类生长因子多肽及

2、其受体分子，通过细胞内信息传递系统刺激细胞增殖。肿瘤的发生与癌基因、抑癌基因和生长因子三者的关系密切相关。第一节 癌基因癌基因（oncogene）就是具有增加癌源性或转化潜能，导致其编码区或调节区域遗传性状发生改变的基因。癌基因可分为两大类：一类是致瘤病毒中能在体内诱发肿瘤并在体外引起细胞转化的基因，即病毒癌基因（viral oncogene, v-onc）；另一类是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，即原癌基因（protooncogene，pro-onc）或称细胞癌基因（cellula

3、r oncogene，c-onc）。一、病毒癌基因和细胞癌基因 （一）概念 癌基因名称用3个斜体小写字母表示，如myc、ras、src。肿瘤病毒是一类能使宿主产生肿瘤或使培养细胞转化成癌细胞的动物病毒。其核酸组成分为DNA病毒和RNA病毒。病毒癌基因是一类存在于肿瘤病毒（大多数是逆转录病毒）中的、能使靶细胞发生恶性转化的基因。 1911年， Reyton Rous 报道将鸡肉瘤的无细胞滤液注射给健康鸡后，可诱导发生肉瘤，表明无细胞滤液含致病原，可传播肿瘤。1932年，Shope发现，野生棉尾兔的皮肤肿瘤也可借助无细胞滤液传播。肿瘤进展：开始，癌细胞处于“休眠”状态，被化学因子、病毒等唤醒后，变

4、的无法无天。Rous提出病毒致癌理论：即传播肿瘤的无细胞滤液中含的是病毒。这种感染性颗粒后来被证实是逆转录病毒(RNA病毒)。Rous因此获得1966年诺贝尔生理和医学奖。癌基因的发现The Nobel Prize in Physiology or Medicine 1966 Tumor-inducing virusesPeyton RousUSARockefeller UniversityNew York, NY, USAB: 1879D:1970鸡肉瘤病毒（RSV)基因组结构图 病毒癌基因与正常细胞中的原癌基因同源 病毒癌基因致癌机制1964年H.M.Temin认为，RSV的生活周期中存在

5、着前病毒（provirus）的DNA中间产物阶段，DNA前病毒含有RNA病毒基因组的全部信息。子代病毒RNA是以前病毒DNA为模版合成的。前病毒可整合到宿主细胞基因组中。通过病毒的诱导，正常细胞可转化成肿瘤细胞。1970年Temin实验室和Baltimove 实验室分别发现RSV病毒粒子中含有反转录酶。这一结果使Temin 的“前病毒”假想得到了证实。1975年Temin、Baltimove和Dulbacco因此获诺贝尔生理和医学奖。The Nobel Prize in Physiology or Medicine 1975 The interaction between tumor viru

6、ses and the genetic material of the cellDavid B BaltimoreUSAMassachusetts Institute ofTechnology (MIT)Cambridge, MA, USAB: 1938Renato DulbeccoUSAImperial Cancer ResearchFund LaboratoryLondon, United KingdomB: 1914 (in Catanzaro, Italy)Howard Martin TeminUSAUniversity of WisconsinMadison, WI, USAB: 1

7、934D: 1994Bishop和Varmus等人于1980年提出了癌基因假说，认为Rous鸡肉瘤病毒的致癌能力与病毒基因组的单个基因有关，即src基因。src基因本是正常细胞基因组的一部分(原癌基因），被病毒“劫持”后，病毒则具有致癌能力。原癌基因在正常细胞中的地位：调控细胞的分裂和生长。肿瘤细胞中癌基因的变化：过分活跃或突变，使其编码产物改变。Bishop和Varmus获1989年诺贝尔生理学和医学奖The Nobel Prize in Physiology or Medicine 1989 The cellular origin of retroviral oncogenesJ. Mic

8、hael BishopUSAUniversity of CaliforniaSchool MedicineSan Francisco, CA, USAB: 1936Harold E. VarmusUSAUniversity of CaliforniaSchool of MedicineSan Francisco, CA, USAB: 1939Press Release: The 1989 Nobel Prize in Physiology or MedicineNOBELFRSAMLINGEN KAROLINSKA INSTITUTETTHE NOBEL ASSEMBLY AT THE KAR

9、OLINSKA INSTITUTE9 October 1989 The Nobel Assembly at Karlinska Institute has today decided to award the Nobel Prize in Physiology or Medicine for 1989 jointly to J. Michael Bishop and Harold E. Varmusfor their discovery of the cellular origin of retroviral oncogenes. Press ReleaseSummaryThe discove

10、ry awarded with this years Nobel Prize in Physiology or Medicine concerns the identification of a large family of genes which control the normal growth and division of cells. Distur-bances in one or some of these so-called oncogenes (Gk nco(s) Bulk , mass) can lead to transformation of a normal cell

11、 into a tumor cell and result in cancer.Michael Bishop and Harold Varmus used an oncogenic retrovirus to identify the growth-controlling oncogenes in normal cells. In 1976 they published the remarkable conclusionthat the oncogene in the virus did not represent a true viral gene but instead was a nor

12、mal cellular gene, which the virus had acquired during replication in the host cell and thereafter carried along. Bishops and Varmus discovery of the cellular origin of retroviral oncogenes has had an extensive influence on the development of our knowledge about mechanisms for tumor development. Unt

13、il now more than 40 different oncogenes have been demonstrated. The discovery has also widened our insight into the complicated signal systems which govern the normal growth of cells. Cellular Oncogenes Discovered by the Use of RetrovirusThe term oncogene was introduced in the middle of the 1960s to

14、 denote special parts of the genetic material of certain viruses. It was believed that this part of the genetic material could direct the transformation of a normal cell into a tumor cell under the influence of other parts of the viral genetic material, alternativelyvia chemical or physical effects.

15、 The favourite theory of the time was that virus-mediated cell-to-cell transmittance of oncogenes was the origin of all forms of cancer. This view was later proven to be incorrect. The original discovery of an oncogenic virus was made in 1916 by Peyton Rous working at the Rockefeller Institute in Ne

16、w York. Fifty years later Rous received the Nobel Prize in Physiology or Medicine. Rous virus, as the infectious agent later was named, is a member of a large virus family named retroviruses. The genetic material of these viruses is RNA (ribonucleic acid). This RNA can be transcribed into DNA (deoxy

17、ribonucleic acid) by a unique enzyme in the virus, reverse transcriptase. The 1975 Nobel Prize in Physiology or Medicine was awarded to David Baltimore, Renato Dulbecco and Haward Temin partly for the discovery of this enzyme. Reverse transcription of the genetic material of the virus into DNA has t

18、he important consequence that it can become integrated into the chromosomal DNA in the cells. It was through investigations of Rous virus that this years laureates Michael Bishop and Harold Varmus in 1975 could demonstrate the true origin of oncogenes. They used one variant of Rous virus which conta

19、ined an oncogenic gene (Figure 1) and another variant which lacked this gene. By use of these viruses they managed to construct a nucleic acid probe which selectively identified the oncogene. This probe was used to search for the corresponding genetic material in DNA from different cells. It was the

20、n found that oncogene-like material could be detected in different species throughout the animal kingdom, in fact even in simple organisms comprising only a few cells. Furthermore, it was shown that the gene had a fixed position in the chromosomes of a certain species, and that the gene, when it con

21、stituted part of the cellular genetic material, was divided into fragments (a mosaic gene) (Figure 1). Figure 1. The difference between an oncogene in a virus and in a cell. In retroviruses causing tumors there is a separate segment of transforming nucleic acid which has been derived from a cell. Th

22、e cellular gene is split (a mosaic gene) whereas the oncogene in the virus is continuous.These findings led to the remarkable conclusion that the oncogene in the virus did not represent a true viral gene but a cellular gene which the virus had picked up far back during its replication in cells and c

23、arried along. This cellular gene was found to have a central function in the cells. It controlled their growth and division. Through these studies of the abnormal, i.e. the diseased state, it was possible to elucidate critical normal cellular functions - a not uncommon situation in biomedical resear

24、ch. The original discovery of a cellular oncogene led to an intensive search for further similar genes. The explosive development of this field of research has led to the identification of more than 40 different oncogenes which direct different events in the complex signal systems that regulate the

25、growth and division of cells. Changes in any one or more of these oncogenes may lead to cancer. Balanced Cellular Interactions - A Biological WonderSymmetrical and asymmetrical, multicellular structures develop from the fertilized ovum by a process of differentiation about which only limited knowled

26、ge is available. In the fully developed individual carefully balanced conditions prevail. Damage of an organ elicits sophisticated repair processes which lead to restitution of the original condition of the organ. However, if a single cell escapes the network of growth control the result may be an a

27、bnormal local proliferation of cells or in the worst case a cancer implying the dissemination of cells running amok. The development of a cancer is a complicated process involving several consecutive changes of the genetic material. Studies of cellular genes (proto-oncogenes) corresponding to the vi

28、ral oncogenes, has started to shed light on the intricate systems which control normal cellular growth and division.Cellular Oncogene Products Constitute Links in Signal Chains which Regulate Growth and Division of Cells The regulation of growth and division of cells has turned out to be much more c

29、omplicated than originally believed. Cellular oncogene products with different properties act in different positions of elaborate signal systems (Figure 2). In order to transmit signals from one cell to the other or from one cell to itself there are growth factors. These factors appear in the fluids

30、 surrounding cells. There are examples of oncogene products, viz. proteins produced in the cytoplasm, which can act as growth factors. Thus, it was found that the product of the sis1) gene was closely related to a previously identified growth factor PDGF (Platelet Derived Growth Factor). Figure 2. O

31、ncogene products are links in signal chains that stretch from the cell surface to the genetic material in the cell nucleus. This chain is composed of (1) growth factors, (2) growth factor receptors, (3) signal transducing proteins in cell membranes, (4) phosphokinases in the cytoplasm and (5) protei

32、ns transported from the cytoplasm into the nucleus where they bind to DNA. The localization of different oncogene products (Sis, ErbB, Ras, Src, Myc) is schematically indicated. In order for a growth factor to be able to interact with a cell there has to be membrane structures, receptors, to which t

33、hey can bind. There are several oncogene products which represent receptors in the cytoplasmic membrane of the cells, e.g. ErbA, Fms, Kit. These receptors have a unique enzymatic activity. They are so-called kinases with a capacity to phosphorylate (=add a phosphate group) the amino acid tyrosine. T

34、here are two more groups of oncogene products with phosphokinase activity; firstly tyrosine/phospho-kinase which lack receptor function and is located at the inside of the cytoplasmic membrane, and secondly serine/threonine phosphokinase which is found in the cytoplasm. Thus, oncogene products funct

35、ion as links in signal chains stretching from the surface of the cell to the genetic material in the nucleus. In the cytoplasm there is one more group of oncogene products. These are called Ras and are related to important cellular signal factors called G-proteins. Finally, there is a large number o

36、f oncogene products which are located in the nucleus of the cell, i.e. Myc, Myb, Fos, ErbA and others. These products direct the transcription of DNA into RNA and therefore play a critical role in the selection of proteins to be synthesized by the cell. Cancer - A Complex, Biological Sequence of Eve

37、ntsChanges in the genetic material constitute the basis for the development of all cancer. Generally there are several consecutive such changes which influence different steps in the signal chains described above. Therefore, one should priori not expect to find one single clue to the mechanism of or

38、igin of cancer. However, application of the expanding knowledge in the oncogene field allows us to start comprehending the disharmonic orchestration behind abnormal cellular growth. It is conceptually incorrect to speak about cancer genes. However,historical circumstances explain why the oncogene te

39、rminology was introduced before a designation of the corresponding normal cellular genes was proposed. From the point of view of cancer the important matter is to compare oncogenes in normal cells and in tumor cells. Oncogenes as a Cause of CancerThe majority of oncogenes have been discovered in exp

40、erimental studies using retroviruses. However, in a few cases oncogenes were identified bythe use of an alternative technique, i.e. genetic material was isolated from tumor cells of non-viral origin and transferred (transfected) to other cells prapagated in culture. The cells receiving the DNA chang

41、ed growth pattern, and further characterization of the transfected genetic material revealed the presence of oncogenes. Two principally different forms of activation of oncogenes can be distinguished. Firstly, the normal cellular oncogene is hyperactive, and secondly the oncogene product is altered

42、so that it can no longer be regulated in a normal way. There are several examples of these types of activation of oncogenes. The discovery of oncogenes was as mentioned originally made by the use of retroviruses. This infers that genetic control elements in the virus itself can be responsible for th

43、e abnormal expression of the oncogene. However, in many cases it was found that alterations of the transferred oncogene contributed to its accentuated expression. There are retroviruses which lack oncogenes but still can induce cancer. This is due to the fact that the virus has inserted its genetic

44、material (in the form of DNA) very close to a normally occurring oncogene in the genetic material of the cell. This may result in an increased turn-over of the oncogene which may lead to abnormal cellular growth. The corresponding phenomenon can also occur in the absence of retroviruses. In this cas

45、e there is a reorgani-zation of the genetic material in the cell. Such a reorganization may occur within a single chromosome or by exchange of material between chromosomes. Repeated copying of a normal oncogene can lead to its amplification in the chromosome and consequently to increased amounts of

46、the oncogene product. In certain brain tumors, glioblastomas, an amplified erbB-gene has been found, and a correspondingly increased neu-gene activity was shown in some forms of breast cancer. The same effect can be seen when there is a reciprocal exchange of segments between chromosomes (translocat

47、ion). Thus the normal myc-gene on chromosome 8 has been translocated to chromosome 14 in many patients with Burkitts lymphomas (Figure 3). The insertion of the myc-gene containing chromosome segment is such that it becomes located close to hyperactive genes directing the synthesis of antibody protei

48、n. As a consequence the myc-gene becomes activated. Chromosome translocations occur in many different tumors. Chromosome analysis can therefore be of considerable value for localization of genetic changes in the genome critical for tumor development. Figure 3. Chromosome translocation in Burkitts ly

49、mphoma. Segments have been exchanged between chromosomes 8 and 14 which has activated the oncogene myc. Oncogenes with point mutations have been observed in many tumors. These mutations may cause alterations in the amino acid composition of the gene product. A well-known example of such a modificati

50、on is the exchange of amino acid 12 from glycine to valine in the ras gene productwhich has been observed in human tumor material. The mutation may also be somewhat more extensive leading to the absence of part of the protein (deletion). Different examples of modified oncogenes in human tumor materi

51、al are given in Table I. The Importance of Viruses for Cancer in ManCancer is not a contagious disease. However, infectious agents like viruses can contribute to the origin of cancer. Thus, it is by use of retroviruses that most oncogenes were identified, the starting materials in such investigation

52、s often being highly specialized, experimentally derived tumors. It seems likely that retroviruses play a relatively limited role for the development of cancer under natural conditions. The only known example in man in which a retrovirus infection contributes to the origin of cancer is the HTLV-1 as

53、sociated lymphomas which occur in Japan. However, there are other kinds of viruses which can contribute to the develop-ment of tumors in man. All these viruses have DNA as their genetic material. As examples can be mentioned papillome (wart) viruses and Epstein-Barr virus, a type of herpes virus. Ce

54、rtain types of papillome viruses play a role for the development of cervical cancer in the genital tract, while Epstein-Barr virus is an important factor for the development of Burkitts lymphomas in Africa and nasopharyngeal cancer in Asia. However, in all these cases factors in addition to the viru

55、s infections are required for the cancer to develop. （二）病毒癌基因和对应原癌基因的比较 原癌基因的限制性表达产物具有促进细胞生长、增殖、分化和发育等生理功能，属于正常的调节基因。细胞原癌基因外显子序列在进化上极为保守，被称为持家基因（house keeping gene），说明这类基因的表达产物在生命活动中是必需的。在受到某些化学、物理或生物性等因素作用下原癌基因因结构、数量等改变而被激活后能使细胞发生恶性转化。 病毒癌基因和对应原癌基因比较有序列的同源性和相似表达产物，但病毒癌基因经过病毒自身改变修饰，和对应的原癌基因比较，主要存在以下

56、一些差别： 1、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。2、病毒癌基因较原癌基因有较强的转化细胞功能， 其原因在于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。3、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。4、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。 （三）癌基因的分类 依据其基因结构与功能特点可将大部分原癌基因归于以下几个家族:1、src家族: 包括src、abl、fes、fgr、fps、fym、kck 、lck、lyn、ros、tkl和yes 等基因。 Src 是最早被发现

57、的癌基因。其表达产物的氨基酸序列具有较高同源性和酪氨酸蛋白激酶活性以及同细胞膜结合的性质，定位于胞膜内侧或跨膜分布。2、ras家族: 包括3类密切相关的成员,即H-ras、K-ras及N-ras。虽其核苷酸序列的同源性较少，但编码蛋白质的分子量均为21 000,即p21。其表达产物多属传递信号的小G蛋白，能结合GTP，有GTP酶活性，并参与细胞内cAMP水平的调节。 3、myc家族: 包括c-myc、L-myc、N-myc、fos、ski等基因,其表达产物定位于细胞核内,为DNA结合蛋白类, 或为转录调控中的反式作用因子,有直接的调节其他基因转录的作用。4、sis家族: 目前认为sis基因仅有

58、一个成员。其表达产物与血小板源生长因子（platelet derived growth factor，PDGF）结构相似，可促进间叶组织的细胞增殖。5、erb家族: 包括erb-A、erb-B、fms、mas、trk等基因,其表达产物是生长因子和蛋白激酶类。6、myb家族: 包括myb和myb-ets复合物等基因,其表达产物为核内转录调节因子，能与DNA结合。 根据表达产物的功能和定位可将原癌基因分为以下几类：1、蛋白激酶类：（1）跨膜生长因子受体 包括erbB、fms、kit、neu（erb-2、HER-2）、ret、sea等基因。（2）膜结合的酪氨酸蛋白激酶 包括src、abl、fes、f

59、gr、fps、fym、kck、lck、lyn、ros、tkl和yes 等基因。（3）可溶性酪氨酸蛋白激酶 包括met、trk等基因。（4）胞浆丝氨酸/苏氨酸蛋白激酶 包括raf（mil、mht）mos、cot、pl-1等基因。（5）非蛋白激酶受体 包括mas、erb等基因。2、信息传递蛋白类：（1）与膜结合的GTP结合蛋白 包括H-ras、K-ras、N-ras等基因。（2）生长因子类 包括sis、int-2等基因。（3）核内转录因子 包括c-myc、N-myc、L-myc、fos、jun等基因。二、癌基因产物的功能 原癌基因表达产物的主要生理功能是调节细胞的生长、增殖、分化，并在细胞内信息传

60、递过程中起到十分重要的作用，这些功能均与正常细胞的增殖密切相关。 (一) 生长因子及其类似物 以生长因子为例。细胞可自分泌（autocrine）生长因子(growth factor, GF)，该生长因子又可促进细胞自身的增殖作用而发生转化。在此过程中，有癌基因产物参与。（二）生长因子受体类 某些原癌基因的表达产物为跨膜受体，主要有两类： 1、酪氨酸激酶类受体 2、非酪氨酸激酶受体 （三）胞内信号转导蛋白类 当胞外生长因子与膜受体结合后，通过胞内一系列传导体（transducer）将生长信号传递到胞内、核内，而引起一系列相关基因的表达而促进细胞增殖。某些癌基因编码参与信号转导激酶级联反应过程的信