Somatic INK4a-ARF locus mutations

A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck

Ming J. Poi, Thomas Yen, Junan Li, Huijuan Song, Jas C. Lang, David E. Schuller, Dennis Keith Pearl, Bruce C. Casto, Ming Daw Tsai, Christopher M. Weghorst

Research output: Contribution to journalArticle

40 Citations (Scopus)

Abstract

The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16INK4a (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14ARF tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G1 arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14ARF genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1α, 1β, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1α. No mutations were found in exon 1β. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14ARF proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin-dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild-type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29- to 38-fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14ARF proteins, seems to be a prevalent event in the development of SCCHN.

Original languageEnglish (US)
Pages (from-to)26-36
Number of pages11
JournalMolecular Carcinogenesis
Volume30
Issue number1
DOIs
StatePublished - Feb 19 2001

Fingerprint

Gene Silencing
Tumor Suppressor Protein p14ARF
Exons
Mutation
Tumor Suppressor Genes
Cyclin-Dependent Kinase Inhibitor p16
Cyclin-Dependent Kinase 4
p16 Genes
Single-Stranded Conformational Polymorphism
Mutant Proteins
Introns
Cyclin-Dependent Kinase 6
Gene Deletion
Human Chromosomes
Codon
Carcinoma, squamous cell of head and neck
Cell Cycle
Nucleotides
Technology
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cancer Research

Cite this

Poi, Ming J. ; Yen, Thomas ; Li, Junan ; Song, Huijuan ; Lang, Jas C. ; Schuller, David E. ; Pearl, Dennis Keith ; Casto, Bruce C. ; Tsai, Ming Daw ; Weghorst, Christopher M. / Somatic INK4a-ARF locus mutations : A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck. In: Molecular Carcinogenesis. 2001 ; Vol. 30, No. 1. pp. 26-36.
@article{8f6932e5e93546c49be649bd22f865f2,
title = "Somatic INK4a-ARF locus mutations: A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck",
abstract = "The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16INK4a (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14ARF tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G1 arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10{\%}, with a combined incidence of 25 of 279, or 9{\%}) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14ARF genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed {"}cold{"} SSCP. Exons 1α, 1β, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27{\%}) exhibited sequence alterations in this locus, 22 (22{\%}) of which were somatic sequence alterations and five (5{\%}) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91{\%}) directly or indirectly involved exon 2, and two (9{\%}) were located within exon 1α. No mutations were found in exon 1β. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14ARF proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41{\%}), a microrearrangement (one of 22, 5{\%}), and single nucleotide substitutions (12 of 22, 56{\%}). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin-dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild-type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29- to 38-fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14ARF proteins, seems to be a prevalent event in the development of SCCHN.",
author = "Poi, {Ming J.} and Thomas Yen and Junan Li and Huijuan Song and Lang, {Jas C.} and Schuller, {David E.} and Pearl, {Dennis Keith} and Casto, {Bruce C.} and Tsai, {Ming Daw} and Weghorst, {Christopher M.}",
year = "2001",
month = "2",
day = "19",
doi = "10.1002/1098-2744(200101)30:1<26::AID-MC1010>3.0.CO;2-E",
language = "English (US)",
volume = "30",
pages = "26--36",
journal = "Molecular Carcinogenesis",
issn = "0899-1987",
publisher = "Wiley-Liss Inc.",
number = "1",

}

Somatic INK4a-ARF locus mutations : A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck. / Poi, Ming J.; Yen, Thomas; Li, Junan; Song, Huijuan; Lang, Jas C.; Schuller, David E.; Pearl, Dennis Keith; Casto, Bruce C.; Tsai, Ming Daw; Weghorst, Christopher M.

In: Molecular Carcinogenesis, Vol. 30, No. 1, 19.02.2001, p. 26-36.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Somatic INK4a-ARF locus mutations

T2 - A significant mechanism of gene inactivation in squamous cell carcinomas of the head and neck

AU - Poi, Ming J.

AU - Yen, Thomas

AU - Li, Junan

AU - Song, Huijuan

AU - Lang, Jas C.

AU - Schuller, David E.

AU - Pearl, Dennis Keith

AU - Casto, Bruce C.

AU - Tsai, Ming Daw

AU - Weghorst, Christopher M.

PY - 2001/2/19

Y1 - 2001/2/19

N2 - The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16INK4a (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14ARF tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G1 arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14ARF genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1α, 1β, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1α. No mutations were found in exon 1β. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14ARF proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin-dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild-type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29- to 38-fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14ARF proteins, seems to be a prevalent event in the development of SCCHN.

AB - The INK4a-ARF locus is located on human chromosome 9p21 and is known to encode two functionally distinct tumor-suppressor genes. The p16INK4a (p16) tumor-suppressor gene product is a negative regulator of cyclin-dependent kinases 4 and 6, which in turn positively regulate progression of mammalian cells through the cell cycle. The p14ARF tumor-suppressor gene product specifically interacts with human double minute 2, leading to the subsequent stabilization of p53 and G1 arrest. Previous investigations analyzing the p16 gene in squamous cell carcinomas of the head and neck (SCCHNs) have suggested the predominate inactivating events to be homozygous gene deletions and hypermethylation of the p16 promoter. Somatic mutational inactivation of p16 has been reported to be low (0-10%, with a combined incidence of 25 of 279, or 9%) and to play only a minor role in the development of SCCHN. The present study examined whether this particular mechanism of INK4a/ARF inactivation, specifically somatic mutation, has been underestimated in SCCHN by determining the mutational status of the p16 and p14ARF genes in 100 primary SCCHNs with the use of polymerase chain reaction technology and a highly sensitive, nonradioactive modification of single-stranded conformational polymorphism (SSCP) analysis termed "cold" SSCP. Exons 1α, 1β, and 2 of INK4a/ARF were amplified using intron-based primers or a combination of intron- and exon-based primers. A total of 27 SCCHNs (27%) exhibited sequence alterations in this locus, 22 (22%) of which were somatic sequence alterations and five (5%) of which were a single polymorphism in codon 148. Of the 22 somatic alterations, 20 (91%) directly or indirectly involved exon 2, and two (9%) were located within exon 1α. No mutations were found in exon 1β. All 22 somatic mutations would be expected to yield altered p16 proteins, but only 15 of them should affect p14ARF proteins. Specific somatic alterations included microdeletions or insertions (nine of 22, 41%), a microrearrangement (one of 22, 5%), and single nucleotide substitutions (12 of 22, 56%). In addition, we analyzed the functional characteristics of seven unique mutant p16 proteins identified in this study by assessing their ability to inhibit cyclin-dependent kinase 4 activity. Six of the seven mutant proteins tested exhibited reduced function compared with wild-type p16, ranging from minor decreases of function (twofold to eightfold) in four samples to total loss of function (29- to 38-fold decrease) in two other samples. Overall, somatic mutation of the INK4a/ARF tumor suppressor locus, resulting in functionally deficient p16 and possibly p14ARF proteins, seems to be a prevalent event in the development of SCCHN.

UR - http://www.scopus.com/inward/record.url?scp=0035145687&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035145687&partnerID=8YFLogxK

U2 - 10.1002/1098-2744(200101)30:1<26::AID-MC1010>3.0.CO;2-E

DO - 10.1002/1098-2744(200101)30:1<26::AID-MC1010>3.0.CO;2-E

M3 - Article

VL - 30

SP - 26

EP - 36

JO - Molecular Carcinogenesis

JF - Molecular Carcinogenesis

SN - 0899-1987

IS - 1

ER -