Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines

Geoffrey I. Shapiro, Julie E. Park, Christian D. Edwards, Li Mao, Adrian Merlo, David Sidransky, Mark E. Ewen, Barrett J. Rollins

Research output: Contribution to journalArticle

Abstract

1 p16INK4A, a specific inhibitor of cyclin-dependent kinase (cdk)4 and cdk6, is a candidate tumor suppressor in malignancies with wild-type retinoblastoma (Rb). Loss of p16INK4A frees these cdks from inhibition, permitting constitutive phosphorylation of Rb and inactivation of its growth suppressive properties. Consistent with this model, Rb-positive non-small cell lung cancers (NSCLCs) have little or no detectable p16INK4A protein, whereas Rb-negative lung cancers have abundant plgiNK4A Howevel.( only some NSCLCs have homozygous deletions or nonsense mutations in a remaining p16INK4A allele, suggesting that other mechanisms must account for absent or low levels of p16INK4A protein. Here, we analyzed 9 Rb-positive NSCLC cell lines for the controls governing p16INK4A activity. Four lines had homozygous deletions p16INK4A (SK-LU-1, SK-MES-1, A-427, and SW900), and three had a point mutation in a single allele. First, in H520 cells, the previously reported deletion at codon 45 results in a frameshift that produces no detectable protein. Second, in Calu-3 cells, a His to Tyr substitution at codon 83 produced a variant with a shortened half-life that was unable to form complexes with cdk4 or cdk6. Third, in H661 cells, the previously reported point mutation in the second intron splice donor site resulted in a smaller p16INK4A protein. Although this variant formed complexes with cdk4 and cdk6, it had a profoundly reduced half-life, producing low steady-state levels of p16INK4A and abundant levels of free cdks. Finally, Calu-1 and Calu-6 cells transcribed no detectable mRNA encoding authentic p16INK4A. These cell lines displayed methylation of the CpG island surrounding the first exon of p16INK4A and expressed abundant levels of a nontranslated mRNA containing an alternative first exon (Elβ), as did all other cell lines in which the p16INK4A locus was not deleted. These data indicate that Rbpositive NSCLC cells have evolved a variety of pathways to suppress p16INK4A expressjon. Reintroduction of p16INK4A into these cell lines by retroviral transfer resulted in a reduced growth rate, increased abundance of hypophosphorylated Rb, accumulation of cells m G1, and a less transformed morphology in Rb-positive, but not Rb-negative cells, suggesting that loss of p16INK4A is essential for maintenance of the transformed phenotype.

Original languageEnglish (US)
Pages (from-to)6200-6209
Number of pages10
JournalCancer Research
Volume55
Issue number24
StatePublished - 1995

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Retinoblastoma
Non-Small Cell Lung Carcinoma
Cell Line
Cyclin-Dependent Kinase Inhibitor p16
Point Mutation
Codon
Half-Life
Exons
Alleles
Cyclin-Dependent Kinase 4
Messenger RNA
RNA Splice Sites
CpG Islands
Nonsense Codon
Sequence Deletion
Growth
Introns
Methylation
Lung Neoplasms
Neoplasms

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Shapiro, G. I., Park, J. E., Edwards, C. D., Mao, L., Merlo, A., Sidransky, D., ... Rollins, B. J. (1995). Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines. Cancer Research, 55(24), 6200-6209.

Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines. / Shapiro, Geoffrey I.; Park, Julie E.; Edwards, Christian D.; Mao, Li; Merlo, Adrian; Sidransky, David; Ewen, Mark E.; Rollins, Barrett J.

In: Cancer Research, Vol. 55, No. 24, 1995, p. 6200-6209.

Research output: Contribution to journalArticle

Shapiro, GI, Park, JE, Edwards, CD, Mao, L, Merlo, A, Sidransky, D, Ewen, ME & Rollins, BJ 1995, 'Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines', Cancer Research, vol. 55, no. 24, pp. 6200-6209.
Shapiro GI, Park JE, Edwards CD, Mao L, Merlo A, Sidransky D et al. Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines. Cancer Research. 1995;55(24):6200-6209.
Shapiro, Geoffrey I. ; Park, Julie E. ; Edwards, Christian D. ; Mao, Li ; Merlo, Adrian ; Sidransky, David ; Ewen, Mark E. ; Rollins, Barrett J. / Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines. In: Cancer Research. 1995 ; Vol. 55, No. 24. pp. 6200-6209.
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AU - Shapiro, Geoffrey I.

AU - Park, Julie E.

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AU - Mao, Li

AU - Merlo, Adrian

AU - Sidransky, David

AU - Ewen, Mark E.

AU - Rollins, Barrett J.

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N2 - 1 p16INK4A, a specific inhibitor of cyclin-dependent kinase (cdk)4 and cdk6, is a candidate tumor suppressor in malignancies with wild-type retinoblastoma (Rb). Loss of p16INK4A frees these cdks from inhibition, permitting constitutive phosphorylation of Rb and inactivation of its growth suppressive properties. Consistent with this model, Rb-positive non-small cell lung cancers (NSCLCs) have little or no detectable p16INK4A protein, whereas Rb-negative lung cancers have abundant plgiNK4A Howevel.( only some NSCLCs have homozygous deletions or nonsense mutations in a remaining p16INK4A allele, suggesting that other mechanisms must account for absent or low levels of p16INK4A protein. Here, we analyzed 9 Rb-positive NSCLC cell lines for the controls governing p16INK4A activity. Four lines had homozygous deletions p16INK4A (SK-LU-1, SK-MES-1, A-427, and SW900), and three had a point mutation in a single allele. First, in H520 cells, the previously reported deletion at codon 45 results in a frameshift that produces no detectable protein. Second, in Calu-3 cells, a His to Tyr substitution at codon 83 produced a variant with a shortened half-life that was unable to form complexes with cdk4 or cdk6. Third, in H661 cells, the previously reported point mutation in the second intron splice donor site resulted in a smaller p16INK4A protein. Although this variant formed complexes with cdk4 and cdk6, it had a profoundly reduced half-life, producing low steady-state levels of p16INK4A and abundant levels of free cdks. Finally, Calu-1 and Calu-6 cells transcribed no detectable mRNA encoding authentic p16INK4A. These cell lines displayed methylation of the CpG island surrounding the first exon of p16INK4A and expressed abundant levels of a nontranslated mRNA containing an alternative first exon (Elβ), as did all other cell lines in which the p16INK4A locus was not deleted. These data indicate that Rbpositive NSCLC cells have evolved a variety of pathways to suppress p16INK4A expressjon. Reintroduction of p16INK4A into these cell lines by retroviral transfer resulted in a reduced growth rate, increased abundance of hypophosphorylated Rb, accumulation of cells m G1, and a less transformed morphology in Rb-positive, but not Rb-negative cells, suggesting that loss of p16INK4A is essential for maintenance of the transformed phenotype.

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