Email: [e-mail]

1
???,??????????????
? ? ? ????????? ??????????? Email
zhuweiguo_at_bjmu.edu.cn
2
?? (Cancer, Tumor, Carcinoma)
Cancer----???? Tumor---?????????
Carcinoma---?????????
Sarcoma----?????????
-oma---???????fibroma,
adenoma (hepatoma,
melanoma????) leukemia
3
Cancer cells
Normal cells
RB
G1
G0
E2F
M
Apoptosis
S
G2
Survival or Death
4
??????????,????,????????????, ?????????????
??
5
??????????
??---???

???????,???, ????, ???????? ?---??
??---????, ?????
6
????????
2500???????
1775?----????Pott ?????, ????????
1918-1933---??,???????????????
??---?? ????---??
?????---??, X? ??
????
7
?????
Oncogene ???
1969?, Huebner RJ, Todaro GJ.
Oncogenes of RNA tumor
viruses as determinants of cancer.
Proc Natl Acad Sci U S A. 1969
Nov64(3)1087-94.
??????????????????????(strange), ????????????????
?
8
What is ???(oncogene)-- ????????????????????????
?????????????????????????????,???????????
,?????????????????????? ???????,????????
????????
9
??????????????,???? ??????????????,????? ?????????
????????? ???????,????????
10
?????????????Src (??Rous??)
Varmus HE, Weiss RA, Friis RR, Levinson W, Bishop
JM. Detection of avian
tumor virus-specific nucleotide sequences in
avian cell
DNAs (reassociation kinetics-RNA tumor
viruses-gas antigen-Rous sarcoma virus,
chick cells). Proc Natl Acad Sci U S A. 1972
Jan69(1)20-4.
Levinson WE, Varmus HE, Garapin AC, Bishop JM.
DNA of Rous sarcoma virus its nature and
significance. Science. 1972 Jan
7175(17)76-8. No abstract available.
????,??????? (proto-oncogene)???,
?????100???????Ras, c-myc, Bcl-2??
11
?????? (Two-hit) 1971?,Knudson?????????????????
????????,????????????????????????,?????,?????????
?????????????????????????? Knudson AG Jr.
Mutation and cancer statistical study of
retinoblastoma.Proc Natl Acad Sci U S A. 1971
Apr68(4)820-3.

12
???????????(Tumor Suppressor Gene, or ????)
??Retinoblastoma, Rb , 1986?

Friend SH, Bernards R, Rogelj S, Weinberg RA,
Rapaport JM, Albert DM, Dryja TP. A human DNA
segment with properties of the gene that
predisposes to retinoblastoma and
osteosarcoma.Nature. 1986 Oct 16-22323(6089)643
-6.
13
????????????????????
Oncogene Tumor Suppressor Genes DNA Repair Genes
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ONCOGENES (???)
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RNA??????????

• ????

DNA??SV40?HPV?EB?????(???????????,?????????) RNA
??(?????)
16
RNA???????

• ???????????????
• 1???????
• Rouse Sarcoma Virus
• ????(??)????????????
• 2???????
• Avian Leukosis Virus
• ?????????????????

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Virus DNA
LTR
gag
pol
env
LTR
Virus protein
Transforming Virus DNA
gag
LTR
LTR
V-onc
Virus protein
?????????????????????
18

• RNA???????????
• ???????????

19

• ?????????????
• RNA?? ???????? ??

????
protein1
protein2
20
?????????

• ????
• 1??????? v-Sis
• 2???????? v-Ros
• 3????????? v-Src
• 4????-????? v-Raf
• 5?RAS?? v-H-Ras, v-K-Ras
• 6????-v-Myc, v-Fos

21

• ??????????????
• 1?????, Int-2
• 2???????, EGFR
• 3???????, Fas
• 4????? Myc
• 5??????? Bcl-2
• 6??????? CDK4

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??DNA?????????
DNA extraction
Tumor Cell
DNA from tumor cells
Screening after transformation
NIH3T3 Cells
Transforming cells
Transforming genes
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????????????? ??????????,????? ????????????????
???
24
??????????

• ???????
• ??????????????????
• H-ras?12????GGC ?? GTC,???????????????,?????p21-R
  as??????????????????

25

• DNA??????
• ????????????????????,??DNA????????????(DMS)???????
  ???????(HSR),?????????????????????????????????????
  ?,??????????????????????????

26
?????????

• DNA?????????????????????,??????????????
• ?????CpG????
• ???????????????????????????????
• ?????????????????,DNA?????????????????,????????

27
?????????

• ???????????????????????????T??????????????????????
  ????????????T?????????????????????????????????????
  B???T?????,??????????????????

28
????????
29
Ras???????

• ??N-ras?H-ras?K-ras
• ??????????,???21000(?????p21)?
• ???,?K-Ras ??, H-Ras, N-Ras????
• Ras?????????????????????????????????????

30

• ???ras???ras????GTP?GDP???????ras????????GTPase
  ???,???????????GTPase????????????ras?GTP??????????
  ??????

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Myc??
??????C-Myc, N-Myc, L-Myc Myc
?????????????????? ?????????????,??Max????????? ??
???????????C-Myc??.
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Bcl-2??????????

• B?????????????,????t(1418) (q32q21)?????
• ???????????????????
• ???25KD
• Bcl-2??????????IL-3??????????IL-3??????????

Bcl-2/Bax ???????????
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FAS-TNF Receptor
Death related protein
Apoptosis proteinase
Bcl-2
Bax
Protein degradation
Cleavage
Damage of cell membrane
Bcl-2????????
34
MDM2?? 12q13-14, 5.5kb mRNA, gene 2.372 kb, AA
491,
MDM2 p53
RB
35
??????

• ??????????????,?????????cyclin??,?????????????(
  cyclin-dependent kinase,CDKs)??????????????????,??
  ???G1???R?,?????????????????????????????????????cy
  clin D,????CDK4??,???????CDK6???????R??

36

• Cyclin D/CDK4????cyclin D?????N?LXCXE???Rb??,?C
  DK4????,?Rb?????,?Rb???????E2F??????????,??????S??
  ?cyclin D??????????????S??,?cyclin/CDK???????????R
  b????

37

• Cyclin D????????30??,????????????,?????????????
  ,?????????cyclin D1?CDK4?G1????,???????????????cyc
  lin D1?????,???G1?????S????,????cyclin
  D??????????????????????????

38

• ?????cyclin D1?cyclin D2????????????

39
(No Transcript)
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???? (Tumor Suppressor Gene, Recessive Oncogene,
Tumor Susceptibility Gene)
41
?????????????????????
1?????????????????? ????? 2???????,????????
?? ????????? 3??????????????????
????,???????????? ??
42
Rb??
Rb(retinoblastoma gene)?????????????
1986?,???????????????????? Rb??,???200kb,?27??
??,26????,?????????31bp,???19kb,???mRNA?4.7kb,??92
8????,????105-110kD?????????Rb??DNA????,??????????
?????
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p15, p18, p19
p57
P57
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??Rb????????
Rb?????????Rb??????????????????Rb????????????G1
?S?????????Rb????????????????????,Rb???????????E2F
??,??????????????????Rb???E2F???E2F?DP1???????????
???G1???S???????????
45
??Rb???????
Rb????????????????????? ??,???????????,?????
??????????????????????13-17?????
???????,???50, ???47, ???32.
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A milestone paper regarding a relationship
between the RB genes and other genes Aberrant
In human cancer cells
Otterson GA, Kratzke RA, Coxon A, Kim YW, Kaye
FJ. Absence of p16INK4 protein is restricted to
the subset of lung cancer lines that retains
wildtype RB.Oncogene. 1994 Nov9(11)3375-8.
47
?????? 1971?,Knudson????????????????????
?????,????????????????????????????,?????,?????????
????????,?????????
48
?????????
1?????????????????? ?????????????????? 2???
??????????????? ????????????
49
p53 ??
?tumor suppressor gene ???,Pubmed???3????,
??????????, ??50????p53???? 1979?,
??SV40???????SV40-large T????, ????????,??1989?,??
???????
50
DeLeo AB, Jay G, Appella E, Dubois GC, Law LW,
Old LJ. Detection of a transformation-related
antigen in chemically induced sarcomas and other
transformed cells of the mouse.Proc Natl Acad
Sci U S A. 1979 May76(5)2420-4.
Baker SJ, Markowitz S, Fearon ER, Willson JK,
Vogelstein B. Suppression of human colorectal
carcinoma cell growth by wild-type p53. Science.
1990 Aug 24249(4971)912-5.
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p53?????
p53????20kb,????????17p13.1,?11??????,??393????
???53kd????????,?????-DNA????-?????????
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p53 protein
??domain 301-393
100-300 AA DNA???
1-99
N
C
53
p53???????
p53 ??????????????,?????????DNA???????????????
?????????
54
p53 ????
p53 ???????????,????????????????
???p53????,????????,????????????????????? ???p53?
???????,???????????
55
p53 ?????????????????????,?????CDK???p21???DNA
?????????????GADD45???p53?G1/S????,??????DNA??,???
????????????????
56
p14arf
MDM2
Many factors
p53
More than 100 proteins
Bax
p21
GADD45
Cell cycle arrest
Apoptosis
57
p53 ???????
p53????????????????????????????
58
???p53??? ???????????????????????????????
?????????5-8????,?????????????????????????????
p53????????????????????,??????????????
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p53????????????????
H273 ?? 249
?? 175 ???
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CDKN2 family
p16ink4a, p15ink4b, p18ink4c and p19ink4d
Cyclin D
Regulate Rb phosphorylation
Cell cycle
61
???????
62
Gene-targeted therapy,?????????, ?????????????????
??????
63
?????
1?1980???????????????????????? 2?80???????????????
????????????? 3?1989?5?22?Rosenberg???????-???????
?????????????????? 4?Anderson?1990?9?4????????????
???,?????????????????????????????????????????,????
???
64
????University of Pennsylvania, ??16????????????
?????????????? 2003?12?,??,????????????
65
????????
?????????????????????????????????????????????????
??? ??? ??? ????
66
???????
?????? ????????? ?????? ex
vivo ?????? in situ ?????? in vivo
67
?????? ex vivo???????????? ???????????????,???? ?
??????????? ?????? in situ???????????? ????????
?,??????,???? ?????? ?????? in
vivo???????????? ?????????
68
???????
????????
???????? 1???????????(?????) 2??????????? 3???????
?????????(??????????)?
69
????????????????? 1?????????
?????????????????,???????????,??????????,???????,?
?????????????????
70
P53 Wildtype-p53-?????
Roth JA. Retrovirus-mediated p53 gene transfer
to tumors of patients with non-small lung
cancer. Nature Medicine, 2985, 1996
No obvious toxicity observed in the retrovirus
28 cases, ????8, ????64 and ??28
Rb, p21, p16, BRCA1
71
2?E1B???????? E1B?????? E1A?E1B?????????????E
1A??Rb?p300????????,?????DNA??,????????E1B?p53????
,?p53????,??????,???????
??E1B??????p53????????p53????,?????????,?????????
?p53???????,??????,?????????????????
72
3???????? ?????????????????????????????? 4???????
?? ?????????????????(HSV-TK)??????,??????ACV?GCV??
???????????,????????????
73
???????
(?)??/???? 1???DNA???????gene gun 2??????????????
? 3??????????? ?????
74
??????
1???????? ??????????????
??,???????Moloney???? ??(Moloney Murine
Leukemia Virus, MoMuLV)?????RNA???
75
Moloney??????
1???RNA?? 2??????????????????,?????????? 3????????
???,?????????gag?pol? env????????????????? 4??????
??????,???????????????
B
A
gag
pol
env
dsDNA
dsRNA
dsRNA
dsRNA
dsRNA
dsDNA
dsRNA
????
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gag
pol
env
???????
????
?????????
???????????
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??Moloney?????????
???????PA317????PA317????????gag?pol?env????????
?????????PA317??,????????????,?????????
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Moloney????????????
??????????????????????????????????????????,??????
? ?????????,??????????????????
??????????????
79
?????????
?????????????????EB???
?????DNA???E1a, E1b ??????????, ???
80
??????
??????????????????????????????????????????????????
????RNA?DNA??,?????????,??????
Ras, Src, Myc????
81
?????????????????? 1??????????????????,??????????
????????RNA? 2???????????????
82
???????????????? 1????????????H???? 2??????5mRNA
????,??mRNA?40s?????? 3???????mRNA???,????????? 4?
???mRNA?????????????? 5???RNA???????????
83
??
?? 1??????RNA? 2???????RNA?
84
RNA INTERFERENCE
Short interfering RNAs lt 30 base pairs
gt 30 base pair double-stranded RNA
Cleavage
2, 5-ASinactive
PKRinactive
RNAi
PKRactive
2, 5-ASactive
Degradation of mRNA (Sequence-specific effects)
(Global effects)
Inhibition of protein synthesis Degradation
of mRNAs
85
DNA ???
X
CmG
methylation
CG
demethylation
86
Thank you for your patience!