Original article
Alterations in the INK4a/ARF locus and their effects on the growth of human osteosarcoma cell lines

https://doi.org/10.1016/S0165-4608(01)00575-1Get rights and content

Abstract

Two different proteins, p16INK4a and p14ARF, encoded by the INK4a/ARF locus play important roles in the RB and p53 pathways, respectively. This study was performed to determine genetic and epigenetic alterations in the INK4a/ARF locus and their effects on the growth of osteosarcoma. Among six cell lines examined, both p16INK4a and p14ARF exons were homozygously deleted in two cell lines, MG63 and HOS, and both p16INK4a and p14ARF promoters were methylated in one cell line, U2OS. Wild-type mRNA and proteins for p16INK4a and p14ARF were expressed in three other cell lines, SaOS2, HuO9, and G292. Transfection studies were performed using two cell lines, U2OS and MG63. Both the RB and p53 genes were wild types in U2OS, whereas p53 but not RB was mutated in MG63. Both p16INK4a and p14ARF suppressed the growth of U2OS, whereas p16INK4a but not p14ARF suppressed the growth of MG63. p53 only did not suppress the growth of MG63 either; however, coexpression of p14ARF with p53 increased the fraction of the G0/G1 phase in MG63 cells. The data presented here demonstrate the importance of genetic and epigenetic alterations in the INK4a/ARF locus for the growth of osteosarcoma and thus will be useful to further understand the biologic behavior of osteosarcoma in association with the defects in the p53 and RB pathways.

Introduction

The INK4a/ARF locus on chromosome 9p21 encodes two distinct proteins, p16INK4a and p14ARF, translated from alternatively spliced mRNAs [1]. p16INK4a is specified by the α transcript comprising exons 1α, 2, and 3 1, 2, 3, 4. The alternative product, p14ARF for “alternative reading frame,” is encoded by the smaller β transcript that comprises exons 1β, 2, and 3 1, 5, 6, 7, 8, 9. The primary amino acid sequences of p16INK4a and p14ARF proteins are completely unrelated because they are produced by translating the common exon 2 sequence in different reading frames 1, 7, 9.

p16INK4a is recognized as a major tumor suppressor implicated in a wide variety of human cancers 1, 10. The fact that transfection of full-length p16INK4a cDNA into p16INK4a−/− cell lines results in marked growth inhibition supports the notion that p16INK4a has a tumor suppressor function [11]. p16INK4a induces G1 arrest by inhibiting the phosphorylation of the RB protein, which is facilitated by the cyclin-dependent kinases cdk4 and cdk6. Thus, ectopic expression of p16INK4a causes cells to be arrested in the G0/G1 phase, but cells that lack the functional RB protein are resistant to this effect 8, 12, 13, 14, 15, 16. p16INK4a is inactivated in cancer cells by several molecular mechanisms, including mutation, deletion, and methylation in the CpG island 1, 17, 18, 19.

p14ARF stabilizes p53 by inhibiting MDM2 and induces cell cycle arrest in both G1 and G2/M 20, 21, 22, 23. p14ARF exon 1β knockout mice developed lymphomas and sarcomas at an early age [24], the phenotype being very similar to that of a previous p16INK4a exon 2 knockout mouse that disrupted both p16INK4a and p14ARF [11]. p14ARF was shown to suppress oncogenic transformation by blocking the ability of MDM2 to mask the transcription-activating function of p53 [25]. In contrast with the results of animal studies, the importance of p14ARF as a tumor suppressor is still unclear in human cancers, because of the reasons hereafter explained. Exons for p14ARF are homozygously deleted in a variety of human cancers; however, it is codeleted with exons for p16INK4a in most cases 26, 27, 28. The p14ARF promoter is known to have methylation in some cancers 28, 29, 30, 31; however, the frequency of p14ARF promoter methylation is still unclear in various types of human cancers. Additionally, to our knowledge, a mutation in exon 1β, unique for p14ARF, has been detected only in a case of human cancer 9, 32, 33, and mutations found in exon 2, common for both p16INK4a and p14ARF, do not appear to be always detrimental to p14ARF activity [34].

Inactivation of both the RB and p53 pathways appears to be essential for the development of osteosarcoma, because either RB or p16INK4a is commonly inactivated together with the p53 gene in osteosarcoma 35, 36, 37. However, the status of the INK4a/ARF locus, in particular of p14ARF, is not fully understood. Thus, we comprehensively examined the status of the INK4a/ARF locus in six osteosarcoma cell lines by analyzing mRNA and protein expression, intragenic mutations and deletions, and promoter methylation of p16INK4a and p14ARF. Furthermore, we investigated the effects of ectopic p16INK4a and p14ARF expression on the growth of osteosarcoma cells without functional p16INK4a or p14ARF.

Section snippets

Cell lines and culture conditions

Six osteosarcoma cell lines were used. SaOS2 was obtained from Dr. R. Takahashi [38]. U2OS was purchased from the American Type Culture Collection (Manassas, VA, USA). HOS, MG63, and G292 were obtained from the Institute for Fermentation (Osaka, Japan) and HuO9 from the Health Science Research Resources Bank (Osaka, Japan). All cell lines, except HuO9, were cultured in DMEM medium containing 10% fetal bovine serum (FBS). HuO9 was cultured in RPMI-1640 medium containing 10% FBS.

Reverse transcription polymerase chain reaction (PCR) analysis of p16INK4a and p14ARF transcripts

cDNA was

Status of p16INK4a and p14ARF in the INK4a/ARF locus in osteosarcoma cell lines

Expression of p16INK4a and p14ARF mRNAs and proteins was examined by RT-PCR and Western blot analyses. We detected mRNA transcripts for p16INK4a and p14ARF in three cell lines, SaOS2, HuO9, and G292, but not in three other cell lines, U2OS, MG63, and HOS (Fig. 1A). Both p16INK4a and p14ARF proteins were detected in SaOS2, HuO9, and G292 but not in U2OS, MG63, or HOS (Fig. 1B). Because the result of RT-PCR analysis was consistent with that of Western blot analysis, it was concluded that both p16

Discussion

In this study, we characterized genetic and epigenetic alterations of p16INK4a and p14ARF in the INK4a/ARF locus of six osteosarcoma cell lines. Both p16INK4a and p14ARF were expressed in three cell lines, SaOS2, HuO9, and G292, and not expressed in the other three cell lines, U2OS, MG63, and HOS. Of the three cell lines without expression, two cell lines, MG63 and HOS, exhibited homozygous deletions of the exons, and one cell line, U2OS, showed promoter methylation. We did not detect mutations

Acknowledgements

This work was supported in part by a Grant-in Aid from the Ministry of Health and Welfare for the Second-Term Comprehensive 10-Year Strategy for Cancer Control. We thank Drs. W.K. Cavenee and T. Kanda for providing us with the pCDKN2WT and pH2B-GFPN1 expression vectors, respectively, and Dr. R. Takahashi for the SaOS2 cell line. Y.-B. Park and M.J. Park are the recipients of an invitation program for foreign researchers from the Foundation for Promotion of Cancer Research. K. Kimura and K.

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