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[Cancer Research 61, 5636-5643, July 15, 2001]
© 2001 American Association for Cancer Research

Tumor Biology

Alterations of p14ARF, p53, and p73 Genes Involved in the E2F-1-mediated Apoptotic Pathways in Non-Small Cell Lung Carcinoma

Siobhan A. Nicholson1, Nader T. Okby1, Mohammed A. Khan, Judith A. Welsh, Mary G. McMenamin, William D. Travis, James R. Jett, Henry D. Tazelaar, Victor Trastek, Peter C. Pairolero, Paul G. Corn, James G. Herman, Lance A. Liotta, Neil E. Caporaso and Curtis C. Harris2

Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892 [S. A. N., M. A. K., J. A. W., M. G. M., L. A. L., N. E. C., C. C. H.]; Orange Pathology Associates, Middleton, New York 10940 [N. T. O.]; Armed Forces Institute of Pathology, Washington, DC 20306 [S. A. N., W. D. T.]; Mayo Clinic, Rochester, Minnesota 55905 [J. R. J., H. D. T., V. T., P. C. P.]; and The Johns Hopkins Oncology Center, Baltimore, Maryland 21231 [P. G. C., J. G. H.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Overexpression of E2F-1 induces apoptosis by both a p14ARF-p53- and a p73-mediated pathway. p14ARF is the alternate tumor suppressor product of the INK4a/ARF locus that is inactivated frequently in lung carcinogenesis. Because p14ARF stabilizes p53, it has been proposed that the loss of p14ARF is functionally equivalent to a p53 mutation. We have tested this hypothesis by examining the genomic status of the unique exon 1ß of p14ARF in 53 human cell lines and 86 primary non-small cell lung carcinomas and correlated this with previously characterized alterations of p53. Homozygous deletions of p14ARF were detected in 12 of 53 (23%) cell lines and 16 of 86 (19%) primary tumors. A single cell line, but no primary tumors, harbored an intragenic mutation. The deletion of p14ARF was inversely correlated with the loss of p53 in the majority of cell lines (P = 0.02), but this relationship was not maintained among primary tumors (P = 0.5). E2F-1 can also induce p73 via a p53-independent apoptotic pathway. Although we did not observe inactivation of p73 by either mutation or DNA methylation, haploinsufficiency of p73 correlated positively with either p14ARF or p53 mutation or both (P = 0.01) in primary non-small cell lung carcinomas. These data are consistent with the current model of p14ARF and p53 interaction as a complex network rather than a simple linear pathway and indicate a possible role for an E2F-1-mediated failsafe, p53-independent, apoptotic pathway involving p73 in human lung carcinogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Rb3 and p53-mediated checkpoints are central to the control of cell cycle progression (1 , 2) . As linchpins of tumor suppression, the disruption of their function is a hallmark of carcinogenesis. These pathways are linked, and their activities are influenced, in turn, by a single genetic locus, INK4a, located on chromosome 9p21 (3 , 4) . This locus encodes two tumor suppressors in a manner unique to eukaryotic cells. p16INK4a is encoded by exons 1{alpha}, 2, and 3, and inhibits the cyclin-dependent kinases 4 and 6. This then inhibits the phosphorylation of Rb, resulting in the sequestration of E2F transcription factors and prevents the transition from G1 to S-phase of the cell cycle (5) . p16INK4a was identified as a tumor suppressor as a result of its association with germ-line mutations in familial melanomas (6, 7, 8) and with frequent deletion, mutation, and promoter methylation in sporadic human tumors and cell lines (9, 10, 11, 12) .

The recently discovered murine p19ARF and its human homologue p14ARF are the alternate transcripts of this locus (13) . p14ARF is encoded by a separate exon 1ß that lies ~20 kb upstream of exon 1{alpha} and shares exons 2 and 3 as read in an ARF, giving rise to a protein completely unrelated to p16INK4a (14) . Despite its unrelated structure, p14ARF also is capable of causing cell cycle arrest in G1 and G2. p14ARF binds to and antagonizes the actions of MDM2, a negative regulator of p53 (15) . Thus, it interferes with the ability of MDM2 to block transcription, to ubiquinate, and to transport p53 to the cytoplasm for degradation (16, 17, 18, 19, 20, 21, 22) . In response to DNA damage, the accumulation of p53 results in cell cycle arrest or apoptosis. The loss of p14ARF increases p53 degradation, thereby diminishing the p53 response to genotoxic stress. The role of p14ARF in carcinogenesis was first demonstrated in mice by showing that ARF-null mice are highly tumor prone (23) and develop sarcomas, lymphomas, carcinomas, and gliomas and thus, die early in life (24) . Mice that are heterozygous for ARF also develop tumors but die later in life. Mouse embryonic fibroblasts that lack ARF do not undergo replicative senescence in culture and can be transformed by oncogenic ras alone (23) . On the basis of this evidence, it was hypothesized that p14ARF functions as a tumor suppressor in vivo.

The modulation of the p53 response to genotoxic stress by p14ARF also raises the possibility that patients, whose tumors show a loss of p14ARF, have a poorer prognosis. This protective role has been demonstrated in mice, where ARF protects cells against Myc-induced tumors, and on an ARF-null background, Eµ-Myc transgenic mice die of aggressive lympholeukemias at an early age (25) . Although alterations of p16INK4a have been demonstrated in ~50% of NSCLCs (26, 27, 28, 29, 30, 31) , the status of p14ARF has not been as well characterized. Although p14ARF deletions are well described, intragenic mutations have not been identified within the unique exon 1ß.

There may be additional associations between p14ARF and other cell cycle regulators, oncoproteins, and tumor suppressors. p14ARF is not activated directly by DNA damage, but rather by a subset of abnormal hyperproliferative signals including oncoproteins myc, ras, and v-abl (32) and E2F-1 (33) . As one of the targets of phosphorylated Rb, the induction of p14ARF by E2F-1 establishes cross-talk between the Rb and p53 pathways (33) . The activities of the E2F family of transcription factors influence both cell cycle progression and apoptosis, and the loss of regulation is seen in many human cancers (34) . In addition to p14ARF, p73, an evolutionary relative of p53 and putative tumor suppressor, also is a downstream target of E2F-1. The activation of p73 has been shown recently to provide a means for E2F-1 to induce cell death in the absence of p53 (35) . Furthermore, the disruption of p73 function inhibits E2F-1-induced apoptosis in p53-null cells (36) . Because E2F-1-induced apoptosis uses both a p53-independent pathway (mediated by p73) and a p53-dependent pathway (mediated by p14ARF; Ref. 37 ), the loss of this apoptotic signal through the disruption of both pathways may enhance tumor development, raising the selection pressure for these genetic alterations in tumors. We tested these hypotheses by studying alterations of exon 1ß of p14ARF and p73 in a series of NSCLCs and sought correlations with other clinical, genetic, pathological, and epidemiological markers examined previously by our group including p53 status. We assessed the prognostic significance of the loss of p14ARF alone or in combination with p53 and p73 in primary-resected tumors. Furthermore, because the p53 and Rb pathways are cross-linked intimately through p14ARF, we also studied p14ARF in a series of human cell lines in conjunction with data acquired previously on the other members of these two pathways including p73, p16INK4a, p53, Rb, and cyclin D1.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Primary Tumor Samples.
DNA extracted from archival NSCLCs and nontumor tissue of 106 patients surgically treated at the Mayo Clinic between 1991 and 1992 formed one arm of this study. An epidemiological profile was available for each patient including demographics, medical, smoking, family, and occupational history, in addition to survival data that now include 9 years of follow-up. The series was composed of 63 males and 43 females, and tumor histology included adenocarcinoma (n = 55), squamous cell carcinoma (n = 29), and large cell carcinoma (n = 22). As part of an ongoing study of these NSCLCs, data were available from previous analyses including p53 mutations and the expression of c-erbB-2 (38) , p21waf1, transforming growth factor-ß1 (39) , bcl-2 (40) , cyclooxygenase-2, NOS2 (41) , LOH in the putative tumor suppressor gene FHIT (42) , and genetic polymorphisms of CYP1A1, CYP2E1, and GSTM1 (43) .

DNA Extraction from Tumor and Nontumor Tissue.
After microdissection of tumor tissue, DNA was isolated by proteinase K and chloroform phenol extraction according to standard protocols (44) .

Cell Lines.
The 53 cell lines analyzed were derived from 11 NSCLCs, 6 SCLCs, 2 mesotheliomas, 11 colon carcinomas, 5 breast carcinomas, 3 pancreatic carcinomas, 5 hepatocellular carcinomas, 2 hepatoblastomas, 1 SV40-immortalized liver cell line, 1 cervical carcinoma line, 1 oral squamous cell carcinoma, 1 lymphoblastic leukemia, 1 lymphoma, 1 glioblastoma, and 1 ovarian carcinoma. We used DNA extracted from stocks of cells frozen at the time of the previous studies (Table 1)Citation .


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  		Table 1 Status of p14ARF, p16INK4a, p53, p73, Rb, and cyclin D1 in cell lines

 
PCR and Sequence Analysis of p14ARF.
Oligonucleotide primers for exon 1ß of p14ARF were synthesized (Bioserve Biotechnologies Ltd., Laurel, MD) according to published sequences. The sequences were 5'-TCCCAGTCTGCAGTTAAGG-3' and 5'-GTCTAAGTCGTTGTAACCCG-3'. The expected 439-bp product includes the 267-bp exon 1ß of p14ARF and 172 bp of noncoding intronic material. Three µl of a DNA template were amplified with AmpliTaq DNA polymerase (Perkin-Elmer) in a total reaction volume of 100 µl that included 10 µl 10x PCR buffer (Perkin-Elmer), 10 µl of DMSO, 2 µl of deoxynucleotide triphosphates, and 20 pmol (2 µl) of each primer. Conditions adapted from Sanchez-Cespedes et al. (45) were 94°C for 5 min and 35 cycles of 94°C for 1 min; 58°C for 1 min; and 72°C for 1 min, followed by a 4-min extension at 72°C on a GeneAmp PCR System 9700 (PE Applied Biosystems). The presence and integrity of DNA was checked by amplifying exons 6 and 7 of p53, which yielded a 150- and 486-bp fragment, respectively. Normal human placental DNA (Sigma Chemical Co., St. Louis, MO) and water included in each reaction served as positive and negative controls, respectively. The amplified 439-bp product was electrophoresed on a 3% agarose gel, visualized by staining with ethidium bromide and UV illumination, and documented by a computer-linked camera. Persistent absence of a band after repeated PCRs with increasing volumes of DNA template, in the presence of a positive result with p53 primers, was interpreted as a homozygous deletion of p14ARF exon 1ß. Seven cases that also failed to amplify the 486-bp product of exon 7 of p53, when known to be present from previous analysis, were excluded from the final series of 86 cases.

The PCR products were column purified (QIAquick PCR Purification kit; Qiagen, Inc., Valencia, CA) and sequenced directly using an ABI PRISM BigDye Terminator Cycle Sequencing kit (PE Applied Biosystems). The same set of primers was used for sequencing and for PCR. The 20-µl reaction volume consisted of 5 µl of PCR product, 2 µl of DMSO, 20 pmol of forward or reverse primer, 3 µl of water, and 8 µl of terminator mix. Reaction conditions were: 25 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. The samples were electrophoresed on an ABI prism 377 DNA Sequencer (PE Applied Biosystems) after a second purification step. The sequence obtained was compared with the known sequence of exon 1ß of p14ARF downloaded from GenBank.

Allelic Deletion Analysis of p73.
Oligonucleotide primers to the chromosome 1p36 gene locus, D1S2893, were retrieved from the Internet site, www.gdb.org, and synthesized. The sequences were 5'-AAAACATCAACTCTCCCCTG-3' and 3'-CTCAAACCCCAATAAGCCTT-5'. Five µl of a DNA template were amplified with AmpliTaq DNA Polymerase (Perkin-Elmer) in a total reaction volume of 100 µl as described above. The conditions were 35 cycles of 94°C for 1 min, 50°C for 1 min, followed by 72°C for 30 s. The 215-bp product was confirmed on a 3% agarose gel. LOH was determined by running each tumor and nontumor amplicon in adjacent lanes on a 5% MetaPhor gel to resolve allelic bands and to identify informative cases. Informative cases with either complete absence or at least 50% attenuation of intensity of allelic bands in tumor samples relative to nontumor samples were considered to have LOH.

SSCP.
The confirmation of LOH was performed by SSCP. Genomic DNA from the LOH pairs was reamplified with [33P]dATP using identical PCR conditions. Sample pairs were heat denatured and run on a 6% polyacrylamide, 5% glycerol gel for 3–5 h at 700 V. The gels were fixed in a 10% acetic acid, 10% methanol solution, and dried and exposed to photographic film for band identification. Cases confirmed by SSCP were subjected to mutational analysis.

PCR and Sequence Analysis of Exons 1–13 of p73.
Samples were analyzed using previously published primers and methods (46) . A modified protocol was used using the Advantage-GC cDNA PCR kit from Clontech. The PCR products were column purified (Concert Rapid PCR Purification System kit; Life Technologies, Inc.). Purified PCR products were sequenced as before using the ABI PRISM BigDye Terminator Cycle Sequencing Reaction kit.

DNA Methylation of p73 Promoter.
The 10 cases that demonstrated LOH of p73 were further analyzed for methylation of the p73 promoter, using a human bacterial artificial chromosome clone containing exon 1 and the 5' region of p73 and methylation-specific PCR. The method has been described previously (47) .

Statistical Analysis.
p14ARF and p73 status within the NSCLC series was correlated with clinicopathological, epidemiological, and survival data acquired previously by our group using a commercially available statistical package, SPSSv10. Associations between the status of p14ARF and p73, and other clinicopathological and genetic markers, were examined using {chi}2 and Fisher exact tests. Associations were considered significant if two-tailed Ps were <0.05. Kaplan-Meier curves plotted cumulative survival against time, and differences in patient survival were analyzed using the log-rank test. Associations between the p14ARF and p53 status and other cell cycle pathway proteins within cell lines also were examined using {chi}2 tests.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
p14ARF Deletions in Cell Lines.
Exon 1ß of p14ARF was deleted from 12 of 53 (23%), mutated in 1 of 53 (2%), and contained WT sequence in the remaining 40 of 53 (75%) cell lines examined (Table 1)Citation . Deletions were present in 4 of 11 (36%) NSCLCs (A2182, A-427, A549, and NCI-H292), 2 of 2 mesothelioma (M24 and M9K), 2 of 3 (67%) pancreatic carcinoma (ASPC-1 and MIA PaCa-2), 1 of 5 (20%) hepatocellular carcinoma (SK-HEP-1), 1 of 1 lymphoma (H9), 1 of 1 glioma (U-118-MG), and 1 of 1 ovarian carcinoma-derived cell line (SK-OV-3). Five of 5 breast carcinomas and 10 of 11 (91%) colon carcinoma-derived cell lines were WT at the genomic level (Fig. 1)Citation . In the colon carcinoma cell line HCT 116, a missense mutation was identified at nucleotide 171 in codon 56, where a G-C bp deletion was found (Fig. 2)Citation .



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  		Fig. 1. PCR demonstration of p14ARF in cell line DNA. A, the 439-bp product is deleted from M9K, A2182, A-427, and A549, but is amplified in 866 MT, Ca Ski, Calu-1, Calu-6, NCI-H1155, and WiDr. B, exon 7 of p53, a 486-bp fragment was amplified as an internal control. The NCI-H358 cell line contains a known deletion of p53. Normal human placental DNA served as a positive control in each reaction.

 


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  		Fig. 2. Sequencing analysis of p14ARF. Nucleotides 160–180 of exon 1ß are illustrated. HCT 116 on the right demonstrates a G-C bp deletion at base 171 compared with a WT sequence on the left.

 
In 13 cell lines with deleted or mutated p14ARF, the p53 status was WT in 9 of 13 (69%) and mutated in 4 of 13 (31%). In 30 of 40 (75%) cell lines WT for p14ARF, p53 abnormalities consisted of mutations in 26, deletions in 4, and p53 was WT in the remaining 10 (25%). Thus, an inverse association was established between p14ARF and p53 in 74% of the cell lines studied (P = 0.002). The correlation was not perfect, however, and p14ARF and p53 were found to be deleted or mutated in 4 cell lines and WT in 10 cell lines (Table 2)Citation .


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  		Table 2 Comparison of p14ARF deletion and p53 status in cell lines

 
The deletion of p14ARF was associated with the deletion of p16 exon 1{alpha} or the loss of p16 protein expression. Although p16 status was not known for every cell line analyzed for p14ARF, in 9 of 13 where p14ARF was deleted or mutated, there also was a deletion or mutation of exon 1{alpha} or a loss of p16 protein expression. One of 13 retained p16 expression, and information was not available for the remaining 3. No association was found with Rb or cyclin D1 status.

Molecular Analysis of Primary NSCLC.
Of the 106 patient total, 86 cases yielded suitable DNA for p14ARF analysis, whereas 87 cases were available for p73 analysis. In 68 cases, data were available for both, but data were only available for either p14ARF or p73 in 18 and 16 cases, respectively.

p14ARF Status in Primary NSCLC.
p14ARF was homozygously deleted from 16 of 86 (19%) NSCLCs and was WT in 70 of 86 (81%; Fig. 3Citation ). No intragenic mutations were found. A statistically significant inverse correlation between p14ARF and p53 status was not observed (P = 0.6). In 7 cases, p14ARF was deleted, and p53 was mutated. p14ARF deletions were almost equally distributed among 7 p53 mutated and 7 WT p53 tumors. The p53 status in the remaining 2 NSCLCs with p14ARF deleted was not available because of technical difficulties (Table 3)Citation . p14ARF deletions were associated with an overexpression of p21waf1 (P = 0.04) and negative staining for bcl-2 (P = 0.05; data not shown). In an examination of prognostic significance, the loss of p14ARF was not associated with a statistically significant shortened 5-year survival for the group as a whole (P = 0.8) nor within the group of WT p53 tumors (P = 0.3).



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  		Fig. 3. PCR demonstration of p14ARF in primary NSCLC. A, a similarly sized fragment, exon 7 of p53, was amplified as a control of DNA quality and integrity. B, detection of p14ARF deletion. p14ARF is deleted apparently from Lanes T1–T6; however, Lanes T4 and T5 are excluded from the final set because they fail to amplify exon 7 of p53. Lanes T1–T3 are interpreted as true homozygous deletions. The 439-bp product is detected in Lanes T7–T9, albeit faintly in Lane T8. C, p14ARF is detected in Lanes T10–T12 and T14, but is deleted from Lanes T13 and T15. Normal human placental DNA served as a positive control in each reaction.

 

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  		Table 3 Comparison of p14ARF deletion and p53 status in primary NSCLCs

 
p73 Status in Primary NSCLC.
There were 48 of 86 (56%) informative cases that demonstrated 2 allelic bands present in nontumor DNA. Ten of 48 (21%) of these informative cases demonstrated LOH in tumor DNA (Fig. 4)Citation . No correlation between p73 and p53 status was observed (P = 0.08). None of these 10 cases with LOH showed evidence of DNA methylation of the promoter region of p73. Sequencing of exons 1–13 revealed WT sequences with no mutations. Five LOH tumor samples (50%) showed a single guanine (G) insertion at nucleotide 778 in the 3' intron of exon 7. The resultant intron did not cause a frameshift mutation or affect the coding region of the gene. Five corresponding normal samples revealed the same insertion, indicating a polymorphism. A single heterozygote A:T to G:C transition was identified at nucleotide 1243 of exon 8. This change was not in the coding region and did not affect the amino acid sequence.



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  		Fig. 4. Demonstration of p73 status by PCR and SSCP. A, 215-bp PCR product of the chromosome 1p36 locus. B, p73 informative cases. Two allelic bands, present in tumor (T) and nontumor (NT), without LOH. C, p73 informative case with LOH. Note 2 allelic NT bands but only one T band. D, SSCP. Note the altered band patterns in the T versus NT sample.

 
Neither the deletion of p14ARF nor p73 LOH correlated with patients’ age, gender, family, smoking, occupational history, tumor histology, size, stage, or grade. We did not detect a statistically significant relationship between either a p14ARF deletion or p73 LOH and other markers studied previously in this series (FHIT LOH; immunohistochemical expression of c-erbB-2, cyclooxygenase-2, NOS2, or genetic polymorphisms of CYP1A1, CYP2E1, and GSTM1). However, we identified a statistically significant association between p73 LOH and p14ARF deletion (P = 0.02; Table 4Citation ). Furthermore, in a three-part analysis of the status of p53, p73, and p14ARF, we noted a statistically significant correlation between p53 mutation, p14ARF deletion, and p73 LOH (P = 0.02). Viewed from another perspective, we demonstrated that p73 LOH occurred only in cases where either p53 or p14ARF, or both together, were mutated or deleted, but not in cases where both were normal (P = 0.01; Table 5Citation ).


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  		Table 4 Comparison of p14ARF and p73 status in primary NSCLCs

 

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  		Table 5 Comparison of p73 status with abnormalities of p14ARF and p53 combined

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have identified p14ARF deletions in a significant number of human cell lines 12 of 53 (25%). Deletions were frequent in NSCLC cell lines (7 of 11), and deletions also were identified in a significant percentage of resected primary NSCLC tumors (19%). Although this figure is <37% cited in an earlier study of resected NSCLCs (45) , these results add support to the premise that p14ARF is a tumor suppressor in lung carcinogenesis. The loss of p14ARF may be more significant in the pathogenesis of NSCLC than SCLC, given that p14ARF deletions in lung cancer cell lines were confined to NSCLC, whereas all 6 SCLC cell lines tested were WT at the genomic level. The high frequency of p53 mutation in SCLC (48) may lessen the selection pressure for p14ARF deletion. Other cell lines with p14ARF deletions in this study were mesothelioma, glioblastoma, and lymphoma derived, which are consistent with previous studies of these tumor types in which alterations of the INK4a/ARF locus have been recognized as frequent events (49, 50, 51, 52, 53, 54, 55, 56, 57) . p14ARF deletions were not identified in colon or breast carcinoma cell lines and in only 1 of 5 (20%) hepatocellular carcinoma lines studied.

A missense mutation identified in exon 1ß of p14ARF of the colon carcinoma cell line HCT 116 demonstrates that an intragenic mutation, not described previously, may occur in exon 1ß of p14ARF, although none were identified in the primary lung tumors studied. HCT 116 is known to harbor a separate mutation in exon 1{alpha} of p16INK4a that results in a stop codon (12) . Thus, in this cell line, both p16INK4a and p14ARF are altered by discreet point mutations. This suggests that although the deletion of p14ARF was associated largely with the deletion of p16INK4a within the group of cell lines studied, abrogation of p16INK4a and p14ARF function may not be governed exclusively by overlapping mutational events.

The subset of cell lines that harbored deletions or mutations of both p53 and p14ARF consisted of a glioblastoma, 2 pancreatic carcinomas, and 1 lymphoblastic lymphoma-derived cell line. These tumor types are noted for their aggressive behavior, suggesting that the loss of both p53 and p14ARF may have additive and cooperative effects in tumorigenesis. It has been shown recently that p14ARF has molecular targets other than p53 (58) , and therefore, the inactivation of p53 may contribute to tumor progression. A report of mice lacking ARF, MDM2, and p53 showed a wider range of tumor types than animals lacking either gene alone. This also was noted in a study of non-Hodgkin’s lymphoma where concomitant alteration of the p53 and the INK4a/ARF locus was associated with a shorter survival (56) .

Nine cell lines and 40 primary tumors were WT for both p53 and p14ARF at the genomic level. Nevertheless, the majority of cell lines in this subset are targeted by mechanisms that act epistatically to disrupt the function of the Rb or p53 pathway. These include the inactivation of p53 by the HPV E6/E7 oncoproteins (Ca Ski; Ref. 59 ) or the T- antigen of SV40 (THLE 5B; Ref. 60 ), the loss of p16 protein expression (LS174T; Ref. 12 ), the deletion of p16INK4a and accumulation of cyclin D1 (MCF7; Ref. 61 ), the loss of Rb (NCI-H146 and NCI-H446), and the methylation of the ARF promoter (RKO; Ref. 62 ). The disruption at this functional level highlights the fundamental significance of the Rb and p53 pathways to carcinogenesis.

Because of the downstream location of p53, the loss of ARF was predicted to be equivalent to p53 inactivation, with the corollary that in any given tumor system the loss of both is functionally redundant and rare (23 , 63) . However, emerging evidence suggests a more complex interrelationship and points to p53-independent functions of p14ARF involving targets other than MDM2 (58) . We found that the correlation of p14ARF with p53 status differed when cell lines, as opposed to primary lung tumors, were examined. An inverse correlation between p53 and p14ARF status was observed among cell lines, which conforms to the earlier model of linear interrelationships between the 2 tumor suppressors. This relationship was not maintained among the primary tumors studied, which echoes the contradictory results reported by others in studies of lung cancer and other tumor types. The evidence that supports the premise of an inverse correlation between p14ARF and p53 is derived from a small number of studies. A statistically significant negative correlation between p14ARF deletions and p53 mutations was established in an analysis of 29 gliomas using a differential PCR method (50) . Acknowledging that this differed from some lung cancer studies, the authors hypothesized that selection pressures for p53 pathway mutagenesis may vary in different tissues. In a study of 97 non-Hodgkin’s lymphomas, ARF deletion was found in 8 cases (9%), mainly in tumors with WT p53, as measured by a multiplex PCR method (56) . In a study of 49 NSCLCs (29) , the loss of expression of ARF protein, as measured immunohistochemically, correlated inversely with p53 overexpression. Normal lung tissue that exhibited nuclear and cytoplasmic staining served as a positive control. Furthermore, the exon 1ß transcript was identified by reverse transcription-PCR in all of a subset of tumors examined. Normal lung also was used as a positive control in a further study of ARF expression in a large group of NSCLCs and high-grade neuroendocrine carcinomas (64) . No relationship was established between p14ARF and p53 status. Reports of immunohistochemical detection of the ARF protein must be interpreted with caution, however, given that the ARF protein is localized primarily in the nucleolus, and extremely low normal levels in adult tissue are detectable only by reverse transcription-PCR (65) . Hence, it is not entirely clear whether the staining pattern observed with rabbit polyclonal antibodies should be interpreted as specific for p14ARF, and whether absence of staining should be regarded as representative of the loss of expression.

In a recent study of 38 NSCLCs, the status of p14ARF was extrapolated from measurements of the LOH by microsatellite analysis. Homozygous deletions were reported in 37% of the cases, and p14ARF inactivation was not inversely correlated with p53 mutation (45) . In a study of colon carcinoma, methylation and inactivation of the ARF promoter was identified in 31 of 110 cases by a methylation-specific PCR. Methylation was overrepresented slightly, but was not restricted to tumors with WT p53 when compared with tumors harboring p53 mutations (62) . We did not address the question of methylation of the ARF promoter in the current study, but it has been demonstrated in 1 of 20 (5%) primary lung cancers.4 We did not establish a negative correlation between p53 and p14ARF in NSCLC at the genomic level, and recognizing the complexity of the cell cycle control network in vivo, we do not consider p14ARF inactivation equivalent to the disruption of the p53 pathway by p53 mutation. This is supported by a recent study of NSCLC that demonstrated multiple impairments of 3 to 4 molecular pathways in 43% of tumors (28) .

We found LOH of p73 in 21% of informative cases, but did not identify intragenic mutations in the expressed allele, which is consistent with the findings of other investigators (66, 67, 68, 69, 70) . However, we did observe a statistically significant positive correlation between p73 LOH and the deletion of p14ARF, which suggests that the reduction of p73 to hemizygosity has functional significance in the dysregulation of E2F-1-induced apoptosis. Because E2F-1 directly activates the transcription of p73, which leads to the activation of p53-responsive target genes that result in apoptosis, and the inhibition of p73 function partially rescues cells from E2F-1-induced apoptosis, thus p73 might constitute a p53-independent, safeguard apoptotic mechanism (37) . It has been shown recently that tumor-derived p53 mutants can bind to and inactivate p73 (71) . Others also have suggested that haploinsufficiency of p73 may lead to dysfunction (72) . Our demonstration of a correlation between p73 LOH and p14ARF deletion in a subset of primary NSCLC is supportive of this hypothesis. Moreover, we propose a model whereby, in the setting of p14ARF loss and/or p53 mutation resulting in a diminution of p53 levels, the "failsafe" p53-independent pathway of apoptosis, mediated by p73, may assume greater significance (Fig. 5)Citation . Should the p73 response be attenuated by mutant p53, haploinsufficiency, or less commonly, by DNA methylation or mutation (46) , then the cell proliferative effects of E2F-1 are favored, tipping the balance toward tumor development. In the current data set, we also have shown that LOH of p73 occurred only where either p14ARF or p53, or both, were abnormal, which suggests that this model of E2F-1 dysregulation is indeed a plausible paradigm of tumor development.



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  		Fig. 5. A model of dysregulation of E2F-1-induced apoptosis. If the apoptotic signals, mediated by p14ARF and p53, are abrogated by deletion, mutation, or DNA methylation, then the p73 safeguard assumes more significance in tumor suppression. However, if p73 function is attenuated by haploinsufficiency, DNA methylation, mutation, or binding to certain mutants of p53, then both apoptotic pathways are inhibited and proliferation is favored.

 
In summary, we have demonstrated that the deletion of exon 1ß of p14ARF occurs in a significant percentage of human cell lines and primary NSCLCs. We also identified LOH of p73 in a similar percentage of lung tumors and a statistically significant correlation with p14ARF deletion and/or p53 mutation that is consistent with the hypothesis about selection pressures for the dysregulation of E2F-1-induced apoptosis in lung carcinogenesis. Furthermore, a statistically significant trend toward inverse correlation between p14ARF and p53 status was seen within the group of cell lines studied, but not within primary tumors. This is consistent with the current model that shows the relationship of p14ARF to p53 as part of a complex network of interactions, rather than a simple linear pathway.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the interviewing and administrative assistance of Marion Barker, RN (Rochester, MN) and the editorial assistance of Dorothea Dudek.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 These authors contributed equally to this work. Back

2 To whom requests for reprints should be addressed, at Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Building 37, Room 2C05, 37 Convent Drive, Bethesda, MD 20892-4255. Phone: (301) 496-2048; Fax: (301) 496-0497; E-mail: Curtis_Harris{at}nih.gov Back

3 The abbreviations used are: Rb, retinoblastoma; ARF, alternative reading frame; MDM, murine double minute; SCLC, small cell lung carcinoma; NSCLC, non-SCLC; LOH, loss of heterozygosity; SSCP, single strand conformation polymorphism; WT, wild type. Back

4 J. G. Herman, unpublished data. Back

Received 1/26/01. Accepted 5/10/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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