Differential inactivation of CDKN2 and Rb protein in non-small-cell and small-cell lung cancer cell lines.
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.


OBJECTIVE
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).


CONCLUSIONS
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.


CONCLUSIONS
Further study of the role of CDKN2 gene alteration in the pathogenesis of NSCLC is needed.
Kelley
M J
MJ
NCI-Navy Medical Oncology Branch, National Naval Medical Center, Bethesda, MD 20889-5105, USA.
Nakagawa
K
K
Steinberg
S M
SM
Mulshine
J L
JL
Kamb
A
A
Johnson
B E
BE
D001483:Base Sequence; D015139:Blotting, Southern; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism*; D018288:Carcinoma, Small Cell / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm / Q000235:genetics; D017353:Gene Deletion; D016147:Genes, Tumor Suppressor; D006720:Homozygote; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012334:RNA, Neoplasm / Q000235:genetics; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D016019:Survival Analysis; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D016160:Retinoblastoma Protein