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Differential inactivation of CDKN2 and Rb protein in non-small-cell and small-cell lung cancer cell lines
M J Kelley1, K Nakagawa, S M Steinberg
1NCI-Navy Medical Oncology Branch, National Naval Medical Center, Bethesda, MD 20889-5105, USA.
Background:
The CDKN2 gene encodes the human cyclin-dependent kinase 4 inhibitor. This inhibitor protein is believed to be a tumor suppressor that plays an essential role in cell cycle regulation. One half of all cancer cell lines and one fourth of lung cancer cell lines examined to date contain homozygous deletions (i.e., both alleles lost) of CDKN2. However, the relative frequency of homozygous CDKN2 deletions in non-small-cell lung cancers (NSCLC) and in small-cell lung cancers (SCLC) has not been determined. Inactivation or loss of another tumor suppressor encoded by the retinoblastoma gene (the Rb protein) is more common in SCLC than in NSCLC.
Purpose:
We measured the frequency of homozygous CDKN2 deletions in 77 NSCLC and in 93 SCLC tumor cell lines. In addition, possible associations were explored between CDKN2 gene loss, the presence or absence of Rb protein, and the clinical status of lung cancer patients.
Methods:
DNA was isolated from each tumor cell line and from the primary tumor and normal tissue of one NSCLC patient. Sequences corresponding to exons 1 and 2 of the CDKN2 gene were amplified by use of the polymerase chain reaction, and the resulting amplification products were analyzed by agarose gel electrophoresis and DNA blotting. Genomic DNA blotting was also used to evaluate CDKN2 gene deletions. The frequency of homozygous CDKN2 loss and the presence or absence of functional Rb protein (reported previously) in the cell lines were compared.
Results:
Homozygous deletion of CDKN2 was detected in 18 (23%) of 77 cell lines established from patients with NSCLC, compared with one (1%) of 93 cell lines established from patients with SCLC (P < .001). No CDKN2 gene loss was observed in the normal tissue of an NSCLC patient whose tumor cell line showed homozygous deletion of the gene; however, the primary tumor from this patient had evidence of CDKN2 loss. Homozygous CDKN2 deletion was detected in 13 (28%) of 46 tumor cell lines from patients with stage III or stage IV NSCLC, compared with zero of 10 tumor cell lines from patients with stage I or stage II NSCLC. Coincident loss of CDKN2 genes and functional Rb protein was rarely observed (in two of 135 cell lines).
Conclusion:
The frequency of homozygous CDKN2 gene deletion in NSCLC cell lines is greater than that observed for any other known, or candidate, tumor suppressor gene.
Implication:
Further study of the role of CDKN2 gene alteration in the pathogenesis of NSCLC is needed.
Insights
Homozygous deletion of the CDKN2 gene, a tumor suppressor, is significantly more frequent in non-small-cell lung cancer (NSCLC) cell lines than in small-cell lung cancer (SCLC) cell lines. This finding highlights CDKN2 alterations in NSCLC pathogenesis.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The CDKN2 gene encodes a tumor suppressor protein crucial for cell cycle regulation.
- Homozygous deletions of CDKN2 are common in various cancer cell lines, but their frequency in specific lung cancer subtypes was unknown.
- Inactivation of the retinoblastoma gene (Rb) is more prevalent in SCLC than NSCLC.
Purpose of the Study:
- To determine the frequency of homozygous CDKN2 deletions in non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) cell lines.
- To explore associations between CDKN2 gene loss, Rb protein status, and patient clinical status.
Main Methods:
- DNA isolation from lung cancer cell lines and patient tissues.
- Polymerase chain reaction (PCR) amplification of CDKN2 exons 1 and 2.
- Agarose gel electrophoresis, DNA blotting, and genomic DNA blotting to assess CDKN2 deletions.
Main Results:
- Homozygous CDKN2 deletion was found in 23% of NSCLC cell lines versus 1% of SCLC cell lines (P < .001).
- CDKN2 loss was observed in advanced stage NSCLC (stages III/IV) but not in early stage (I/II).
- Coincident loss of CDKN2 and functional Rb protein was rare.
Conclusions:
- The frequency of homozygous CDKN2 deletion is higher in NSCLC cell lines than any other known tumor suppressor gene.
- Further investigation into CDKN2 alterations in NSCLC pathogenesis is warranted.