PMID- 10389768
OWN - NLM
STAT- MEDLINE
DCOM- 19990719
LR  - 20190708
IS  - 0020-7136 (Print)
IS  - 0020-7136 (Linking)
VI  - 82
IP  - 2
DP  - 1999 Jul 19
TI  - Functional reassessment of P16 variants using a transfection-based assay.
PG  - 305-12
AB  - CDKN2A appears to be the major melanoma susceptibility gene, and is also
      mutated/deleted in sporadic tumours of various types including melanoma. Thus far
      most approaches to assessing the functionality of mutations in this gene have
      used in vitro methods such as CDK4 binding and kinase inhibition assays, with
      sometimes disparate conclusions about functional significance of some variants
      between studies. We have used a melanoma cell line (MM96L) with no functional
      p16, as the basis for a "semi-in vivo" transfection-based assay for exogenous p16
      functionality based on the growth parameters of the cells and the behaviour of
      variant proteins after transfection of different CDKN2A cDNAs. Colony counts
      performed on these transfectants revealed that all but the wild type, + 24 bp ad 
      A148T variants have a diminished ability to inhibit cell growth. All other
      variants detected either constitutionally in familial melanoma patients (I49T,
      R87P, G101W and V126D) or somatically in melanomas (N71S, and P81L), appeared
      functionally impaired in this assay. This diminution of function was independent 
      of CDK4 and CDK6 binding ability. Furthermore, the predominant localization of
      these variants within the cell was different from that of wt p16. This
      mislocalization may provide an explanation for their lack of function, or
      alternatively, it may also be an indicator that the cells are processing
      unstable, misfolded p16 proteins. This novel assay for assessment of
      functionality of p16 variants may better reflect the role of some of these
      mutations in vivo, and as such is a useful adjunct to other in vitro assays.
FAU - Walker, G J
AU  - Walker GJ
AD  - Joint Experimental Oncology Program, Queensland Institute of Medical Research,
      Royal Brisbane Hospital, Herston, Australia.
FAU - Gabrielli, B G
AU  - Gabrielli BG
FAU - Castellano, M
AU  - Castellano M
FAU - Hayward, N K
AU  - Hayward NK
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Int J Cancer
JT  - International journal of cancer
JID - 0042124
RN  - 0 (Cyclin-Dependent Kinase Inhibitor p16)
RN  - 0 (DNA, Complementary)
RN  - 0 (DNA, Neoplasm)
RN  - 0 (Neoplasm Proteins)
RN  - 0 (Recombinant Fusion Proteins)
SB  - IM
MH  - Cell Nucleus/chemistry
MH  - Cyclin-Dependent Kinase Inhibitor p16/*deficiency/genetics/physiology
MH  - Cytoplasm/chemistry
MH  - DNA, Complementary/genetics
MH  - DNA, Neoplasm/genetics
MH  - Fluorescent Antibody Technique, Indirect
MH  - Gene Deletion
MH  - *Genes, p16
MH  - Genetic Predisposition to Disease
MH  - Genetic Variation
MH  - Humans
MH  - Melanoma/*genetics
MH  - Mutation
MH  - Neoplasm Proteins/*deficiency/genetics/physiology
MH  - Polymorphism, Genetic
MH  - Recombinant Fusion Proteins/physiology
MH  - Reverse Transcriptase Polymerase Chain Reaction
MH  - Transfection
MH  - Tumor Cells, Cultured
MH  - Tumor Stem Cell Assay
EDAT- 1999/07/02 10:00
MHDA- 2000/06/20 09:00
CRDT- 1999/07/02 10:00
PHST- 1999/07/02 10:00 [pubmed]
PHST- 2000/06/20 09:00 [medline]
PHST- 1999/07/02 10:00 [entrez]
AID - 10.1002/(SICI)1097-0215(19990719)82:2<305::AID-IJC24>3.0.CO;2-Z [pii]
AID - 10.1002/(sici)1097-0215(19990719)82:2<305::aid-ijc24>3.0.co;2-z [doi]
PST - ppublish
SO  - Int J Cancer. 1999 Jul 19;82(2):305-12. doi:
      10.1002/(sici)1097-0215(19990719)82:2<305::aid-ijc24>3.0.co;2-z.