Molecular BIology of Cancer Topics             

p53 and Cdk Inhibitors

p53 tumor supressor protein is a transcription factor. The protein contains several important domains: DNA binding domain, transcriptional transactivation domain, and conserved domains I - V. The extreme carboxy terminus of the p53 protein contains several nuclear localization sequences (NLSs) and phosphorylation sites.

The products of genes transactivated by p53 include cdk inhibitors (p21, WAF1, Cip1), the p53 inhibitor MDM2, GADD45 (induced by DNA damage, involved in excision repair), cycling G (unknown function), the proapoptotic portein Bax, and IGF-BP3 (blocks signaling of mitogenic growth factor).

As shown by double-knockout mice, p53 is not required for development, but lack of it predisposes the animals to cancer, especially sarcomas and lymphomas.

p53 gene mutations occur in combination with 17p allelic deletions (LOH) as late events in colorectal tumorigenesis (Baker et al. Cancer Res. 50:7717-7722, 1990), i.e. in the progression stage of the cancer. There are fewer p53 mutations in benign adenomas than in malignant carcinomas. loss of heterozygosity (LOH) is more frequent in carcinomas than in adenomas. Most carcinomas with a p53 missense mutation also exhibit LOH.

Loss of p53 accelerates progression from benign to malignant tumors. Lack of p53 is associated with rapid progression from bening to malignant neoplasms.

Oncogenic Mutant p53

Although wild-type p53 has tumor suppressor activity, mutant p53 does not. In an experiment were the p53 negative cancer cell line SAOS-2 was transfected with either wild-type or mutant p53 genes, only the cells transfected with the wild-type gene were prevented from growing in soft agar and causing tumors in athymic (immunocompromized) mice (Chen et al. Science 250: 1576-1580, 1990). Since p53 must for homodimers to be functional as transcription factors, mutant p53 inhibits transcriptional transactivation by wild-type p53 (i.e one mutant allele has a dominant-negative effect). Because p53 functions as an homodimer, mutant p53 can be thought of as an oncogene.

p53 is often mutated in cancer, with missense mutations at many different codons. 98% of p53 missense mutations occur in conserved domains II-V, e.g. Arg (R) 248. The most frequent p53 mutations in human cancers occur in codons 248 and 273 which encode arginines that bind DNA, thus preventing DNA binding. p53 mutations are present in nearly every cancer type, and p53 is the most frequently mutated gene in human cancer. Li-Fraumeni syndrome is due to an inherited missense mutation in p53.

Protein Interactions

Many proteins that interact with p53 act as oncogenes when viral or mutated by inhibiting p53 function. The viral proteins SV40 T antigen and AdE1B blocks p53 DNA binding domain and transcriptional activation domain, respectively. The human papilloma virus E6 promotes degradation of p53; this occurs in most cervical cancers.

A mutant adenovirus has been developed that selectively kills cells lacking wild-type p53 function (Science 274: 373-378, 1996). Because it lacks the E1B protein that shuts down wild-type p53, the mutant dl1520 adenovirus cannot reproduce in wild-type p53-bearing cells, but reproduces and kills cancer cells without wild-type p53 function.

Oncogenic mdm-2 and c-Abl blocks p53 transcriptional activation domain and p53-mediated cell arrest, respectively. Mdm-2 is amplified in human sarcomas. The mdm-2 gene was cloned from mouse double-minute chromosomal material. Mdm-2 protein (MDM2) coprecipitates with p53 (wild type and mutant) in mamalian cells.

DNA Damage Response

p53 is induced in response to DNA damage, for example by X-rays, resulting in G1 cell growth arrest while DNA is repaired. In normal cells, MDM2 is bound to wild-type p53 and may target p53 for degradation. In DNA damaged cells, wild-type p53 is phosphorylated and thus freed of MDM2. Free p53 can activate transcription of target genes.

There are two possible fates of DNA-damaged cells with increased wild-type p53: G1 arrest and DNA damage repair or apoptosis. A known mechanism of tumor suppression by p53 involves transcriptional activation of p21/Waf1/Cip1. p21 protein levels increase in response to wild-type but not mutant p53. p21 binds to and inactivates cdk2-cyclin complexes, thus arresting the cell cycle.

Mutant p53 fails to respond to DNA damage. In the absence of wild type p53, cells continuer to divide even with serious DNA damage, resulting in further mutations and aneuploidy, thus leading to tumor progression. Wild-type p53 is also necessary for DNA damage induced apoptosis. Cells with irreparable DNA damage cannot undergo apoptosis when laking wild type p53.

Many DNA damaging drugs cause p53 accumulation: cisplatin (DNA croslinker), mitomycin C (alkylating agent), etoposide (vinca alkaloid), 5-fluroureacil (nucleoside analog) and others.

p16 and Other Cdk Inhibitors

p16 is another cyclin/cdk inhibitor that may function as a human tumor suppresor. It prevents G1/S progression by inhibiting the cdk4/cyclinD complex. Deletion of its gene at segment 9q21 is found in families with an inherited predisposition to melanoma, and is deleted in some cancer cells. Abrogation of the Rb/p16 tumor suppressive pathway is found in virtually all pancreatic carcinomas, and may be due to mutantation and LOH, deletion and LOH or methylation/decreased expression.

Other cdk inhibitors include Far1 (G1 arrest in yeast) and p27 (G1 arrest in mammals). p27 is activated in response to the atimitogen TGF-b.

Continue to "DNA Repair" or take a quiz: [Q1].

Need more practice? Answer the review questions below.

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1- What is p53?
p53 tumor suppressor protein is a transcription factor that transactivate expression of cell cycle inhibitors, proteins involved in DNA repair, and proapoptotic proteins.

2- List 5 important domains of the p53 protein.
DNA binding domain
transcriptional transactivation domain
conserved domains I - V
nuclear localization signal at the extream carboxy terminus
phosphorylation sites

3- List 8 proteins induced by p53 and their function.
p21, cdk inhibitor
WAF1, cdk inhibitor
Cip1, cdk inhibitor
MDM2, p53 inhibitor
GADD45, excision repair
cyclin G, unknown function
Bax, proapoptotic protein
IGF-BP3, blocks signaling of mitogenic growth factor

4- What has been revealed by double-knockout p53 mice experiments?
p53 is not required for development but lack of it predisposes the animal to cancer, especially sarcomas and lymphomas.

5- Which p53 genetic changes occur in late colonorectal cancers?
Mutations and 17p allelic deletions (LOH).

6- What happens to tumors when p53 is lost?
Loss of p53 accelerates progression from bening to malignant tumors.

7- Is mutant p53 a tumor supressor gene? Explain using experimental data.
No. WHen the p53-negative cell line SAOS2 is transfected with either wild type or mutant p53l, only the wild-type transfected cells are prevented from growing in soft agar and causing tumors in athymic mice.

8- Why is mutant p53 not a tumor suppressor gene?
Since p53 must form homodimers to be functional as transcription factor, mutant p53 inhibits transcriptional activation by wild-type protein, i.e. has a dominant--negative effect. Thus mutant p53 can be thought of as an oncogene.

9- Which are the most common p53 mutations in cancer?
Missense mutations at many different codons may occur, 98% in conserved domains II-V, e.g. Arg (R) 248. The most common mutations in human cancers occur in codons 248 and 273 which encode arginines that bind DNA, thgus preventing DNA binding.

10- Which cancer types have p53 mutations?
p53 mutations are present in nearly all cancer types, and p53 is the most frequently mutated gene in human cancer. Li-Fraumeny syndrome is due to an inherited missense mutation in p53.

11- Which oncogenic viral proteins interact with p53 and how?
SV40 T antigen and adenovirus E1B block p53's DNA binding and transactivation domains. Human papilloma virus E6 promotes degradation of p53 in most cervical cancers.

12- Explain how a mutant adenovirus can be used to kill cancer cells.
A mutant adenovirus has been developed that selectivelly kills cells lacking wild-type p53 function. Because it lacks the E1B protein that shuts down wild-type p53, the mutant adenovirus cannot reproduce in cells wit wild0type p53, but reproduces and kills cancer cells without the wild-type p53.

13- Which oncogenic non-viral proteins interact with p53 and how?
Oncogenic mdm-2 blocks p53 transcriptional activation domain. Oncogenic c-Abl blocks p53-mediated cell cycle arrest.

14- What genetic change of mdm-2 gene can be found in human sarcomas?
Mdm-2 is amplified in human sarcomas.

15- How was the mdm-2 gene discovered?
It was cloned from mouse double-minute chromosomal material.

16- What is the function of mdm-2?
In normal cells, mdm-2 is bound to wild-type p53 and may target p53 for degradation.

17- What is the relation between p53, mdm-2 and DNA damage?
p53 is induced in response to DNA damage, for example by X-rays, resulting in G1 arrest while DNA is repaired. In DNA-damaged cells, wild-type p53 is phosphorylated and thus freeed from mdm-2. Free p53 can activate transcription of target genes.

18- What are the 2 possible fates of cells with DNA damage and increasing levels of free p53?
G1 arrest and DNA repair
Apoptosis

19- How do p21 works?
After active p53 increases p21 expression, p21 binds to and inactivates cdk2-cyclin complexes, thus arresting the cell cycle.

20- How do mutant p53 works when there is DNA damage.
Fails to respond and activate transcription of genes that would otherwise arrest the cell cycle or induce apoptosis.

21- What are the consequences of nonfunctional p53 after DNA damage?
Cells continue to divide even with serious DNA damage, resulting in further mutations and aneuploidy. Abnormal cells are not able to undergo apoptosis, thus leading to tumor progression.

22- How can some chemotherapeutic drugs exploit p53 abilities as tumor suppresor?
They damage DNA, thus causing p53 accumulation.

23- List 5 DNA damaging drugs that cause p53 accumulation. Bonus: list mechanism of action.
cisplatin (DNA croslinker)
mitomycin C (alkylating agent)
etoposide (topoisomerase inhibitor)
5-flurouracil (nucleoside analog)

24- What is p16?
p16 is another cyclin/cdk inhibitor that may function as a human tumor suppressor gene. I prevents G1/S progression by inhibiting the cdk4-cyclinD complex.

25- Which cancers are associated with non-functional p16?
Deletion of the p16 gene at segment 9q21 is found in families withh predisposition to melanoma, and isdeleted from some other cancer cells. Abrogation of the Rb/p16 tumor supressive pathway is found in virtually all pancreatic carcinomas.

26- List 3 genetic changes of the Rb/p16 pathway in pancreatic carcinoma.
mutation and loss of heterocygocity (LOH)
deletion and LOH
methylation/decreased expresion.

27- List 4 cdk inhibitors.
p21
p16
Far1
p27.

28- What are the mechanisms of action of Far1 and p27?
Far1 causes Gi arrest in yeast by inhibiting cdk. p27causes G1 arrest in mammals by inhibiting cdk, in response to the antimitogen TGF-b.