conditional knockout of p53 in mesenchymal cells of mice results in osteosarcomas
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CONDITIONAL KNOCKOUT OF P53 IN MESENCHYMAL CELLS OF MICE RESULTS IN OSTEOSARCOMAS. PATRICK P. LIN, FENGHUA JIN, GUILLERMINO LOZANO November 13, 2004 Montreal, Canada. Introduction. P53. Tumor suppressor gene Most commonly mutated tumor suppressor Approximately 50% of all cancers - PowerPoint PPT PresentationTRANSCRIPT
CONDITIONAL KNOCKOUT OF P53 IN MESENCHYMAL CELLS OF MICE
RESULTS IN OSTEOSARCOMAS
PATRICK P. LIN, FENGHUA JIN, GUILLERMINO LOZANO
November 13, 2004Montreal, Canada
Introduction
P53
• Tumor suppressor gene– Most commonly mutated tumor suppressor – Approximately 50% of all cancers
• Guardian of the genome– Activated in response to DNA damage– Cell cycle arrest– Apoptosis
Li-Fraumeni SyndromeGerm-Line P53 Mutation
Hisada, JNCI 90:606, 1998
P53 Knockout Mice
• Developed in early 1990s
• Mice exhibit a wide spectrum of tumors
• Problem – Homozygous knockout die of lymphomas rapidly
• Within 6 months
• Relatively few sarcomas
– Heterozygous knockout produces sarcomas slowly• Usually about 18 months
P53 Deficient Mice
Heterozygousknockout
Homozygousknockout
Jacks et al, Current Biology 4:1, 1994Donehower, Current Biology 7:296, 1996
Rb
• Another important tumor suppressor gene
• Involved in cell cycle control
Hereditary RetinoblastomaGermline Mutation of Rb
• Osteosarcoma 2nd most common malignancy
• Rb - mutations found in sporadic osteosarcomas also
Rb Knockout Mice
• Homozygous mice are embryonic lethal
• Heterozygous mice only develop pituitary tumors– No osteosarcomas or other sarcomas
Goal of This Study
• Develop genetic mouse model of sarcomas– Effect of specific mutations on tumor phenotype
• Tumor suppressor genes
Strategy
• Conditional knockout of tumor suppressor genes – Early knockout
• Less differentiated cells more likely to give rise to tumors
– Only in mesenchymal tissue • Bone, cartilage, muscle, connective tissue
Cre-Lox Recombination
• Cre recombinase– Transgenic mouse expresses Cre under the control of a specific
promoter
• LoxP sites– Short 34 bp sequence recognized by Cre
– Introduce into mouse genome around targeted gene
creloxP loxP loxP
gene
Published WorkRb/P53 Conditional Knockout in Brain
• GFAP-Cre x P53lox/lox Rblox/lox
– 100% medulloblastomas • within 4 months
• GFAP (glial fibrillary acidic protein) – expressed only in brain
Marino et al, Genes & Dev 14:994, 2000
Prx1
• Paired-related homeobox gene– Previously called Mhox– Regulates embryonic development
• Prx1 Knockout mice– Craniofacial defects– Limb shortening– Spina bifida
Prx1 Expression in Embryoes
• Primarily in Limbs & Cranial Mesenchyme
Martin, Genesis 26:225, 2000
Prx1 Expression in Embryonic Tissue
• Not in epithelium – (e & ep, panels B,D)
• Only in mesoderm– Condensing mesenchyme
of limb bud (cm, panel C)
– Mesenchyme (m, panel D)
– Periosteum (p, panels E,F)
– Maxillary process (mp, panels A,B)
Martin, Genesis 26:225, 2000
Prx1-Cre Transgenic Mouse
• Utilizes 2.4 kb fragment– 5’ genomic flanking region of Prx1 gene– contains 530 bp core fragment of Prx1 promoter
Logan, Genesis 33:77, 2002
Prx1-Cre Expression
• Prx1-Cre is expressed primarily in limbs– Cross to R26R (Rosa26 lacZ reporter)
P53lox and Rblox Mice
loxlox
1 2 3 4 5 6 7 8 9 10 11
P53lox
Rblox
Exon 19
loxlox
Marino, Genes & Dev 14:994, 2000
Results
Tumor Cohorts
Cohort #Mice Age (wks)
Tumors
Prx1-Cre+/- Rblox/lox 13 44-98 1
Prx1-Cre+/- Rblox/NULL 8 44-98 0
Prx1-Cre+/- P53lox/WT 23 33-96 4
Prx1-Cre+/- P53lox/lox 25 42-68 18
Prx1Cre – mediated knockout of p53
Time (wks)
120100806040200
Ove
rall
Su
rviv
al
1.0
.8
.6
.4
.2
0.0
p53-lox/lox
p53-lox/w t
p=0.008
Cause of DeathPrx1cre P53 lox/lox
Osteosarcoma
Soft tissue sarcoma
Non-tumor
Lymphoma
Osteosarcomamouse 1296 age 39 wks
femur
Location of Osteosarcoma Proximal Femur Predominant Site
Mouse 1304age 40 wks
scapula
Genotype of Tumor
• PCR verifies loss of floxed P53 gene in tumor
Tail
Tumo
rNeg
contr
ol
Osteoblastic Osteosarcoma
Osteosarcoma Metastasis Prx1cre P53 lox/lox
• Lung: 2 of 12 (17%) mice– Visible on Xray or necropsy
– Microscopic mets not easily detectable
– Primary tumor grows extremely fast (days)
• Bone: 4 of 12 tumors• Spleen: 1 of 12 tumors
Metastatic vs. Multifocal Disease?
• 4 of 12 (33%) mice • Osteoblastic tumors in
multiple bones• Multifocal disease vs. mets?
– 1 mouse had both lung and bone mets
Soft Tissue Sarcoma
• Poorly differentiated, unclassified soft tissue sarcoma
• No osteoid
• No involvement of bone
Lymphoma of Bone
• Rarely seen• Arose in bone• Thymus not involved
Rb Conditional KnockoutRblox/lox and Rblox/null
• No developmental abnormalities– Limbs normal
– Mice fertile
• No sarcomas– Most die of old age
– 1 tumor (thyroid)
• Rb knockout does not initiate sarcoma formation in mice! Time (wks)
100806040200
Ove
rall
Su
rviv
al
1.0
.8
.6
.4
.2
0.0
Rb lox/null
Rb lox/lox
Rb Knockout Accelerates Sarcoma Formation
• Simultaneous conditional knockout of p53 and Rb
• p < 0.0001
• Preliminary data
– Utilizes col2A1cre mouse to achieve double conditional knockout
Weeks
80706050403020100
Su
rviv
al
1.0
.8
.6
.4
.2
0.0
col2A1crep53lox/lox Rblox/lox
prx1crep53lox/lox
Weeks
80706050403020100
Su
rviv
al
1.0
.8
.6
.4
.2
0.0
col2A1crep53lox/lox Rblox/lox
prx1crep53lox/lox
Discussion
Preliminary Study
• Establishes framework for future studies
• Larger numbers of mice needed to corroborate initial findings here
Mice Tend to Make Osteosarcomas
• Distribution of tumors is not random– Histology - osteosarcomas– Location - femur – Not predicted
• Prx1-cre knock outs p53 in the limb bud• All mesenchymal tissues in limb should have equal chance of tumor
formation
• There must be strong genetic & developmental influences that favors osteosarcoma in femur
P53 Conditional Knockout
• Good strategy to create sarcomas– Eliminate nearly all other tumors
• Occasional lymphoma of bone occurs
– Confirms hypothesis that mice bearing homozygous deletion of p53 will almost always develop sarcomas
• P53 mutation is an initiating event for sarcomas– Represents one important pathway for sarcoma formation
– This is generally not believed to be true for carcinomas
Rb Conditional Knockout
• No developmental defects (surprisingly)– Note global Rb knockout is lethal in embryoes
• Poor initiator of sarcoma formation in mice– No tumors with Rb knockout alone
• Accelerates sarcoma formation– Double knockout of p53 & Rb produces sarcomas
more rapidly
Conclusions
• Conditional knockout of p53 produces sarcomas – Majority are osteoblastic osteosarcomas of femur– Poorly differentiated soft tissue sarcomas also form
• Conditional knockout of Rb accelerates sarcoma formation– Does not initiate sarcoma formation in mice
Acknowledgements
• Orthopaedic Research & Education Foundation Grant (02-026)
• Anton Berns• John Parant• Jim Martin• Carolyn Van Pelt• Richard Behringer• Victor Olnichikov
Thank You