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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, 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