University of Massachusetts Medical School eScholarship@UMMS Open Access Articles Open Access Publications by UMMS Authors 1996-03-01 Gene deletion chemoselectivity: codeletion of the genes for p16(INK4), methylthioadenosine phosphorylase, and the alpha- and beta-interferons in human pancreatic cell carcinoma lines and its implications for chemotherapy Zhi-Hao Chen University of Massachusetts Medical School Et al. Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/oapubs Part of the Cancer Biology Commons, and the Oncology Commons Repository Citation Chen Z, Zhang H, Savarese TM. (1996). Gene deletion chemoselectivity: codeletion of the genes for p16(INK4), methylthioadenosine phosphorylase, and the alpha- and beta-interferons in human pancreatic cell carcinoma lines and its implications for chemotherapy. Open Access Articles. Retrieved from https://escholarship.umassmed.edu/oapubs/371 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Open Access Articles by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. [CANCERRESEARCH56, 1083-1090, March1, 19961 Gene Deletion Chemoselectivity: Codeletion of the Genes for p16@'@4, Methylthioadenosine Phosphorylase, and the a- and @-Interferons in Human Pancreatic Cell Carcinoma Lines and Its Implications for Chemotherapy' Zhi-Hao Chen, Hongyang Zhang, and Todd M. Savarese2 Cancer Center, University of Massachusetts Medical Center, Worcester, Massachusetts 01655 ABSTRACT Loss of these “innocentbystander―genes may be significant, because loss of the expression of the proteins encoded by these genes may alter Pancreatic carcinoma cells lines are known to have a high incidence of cellular physiology and, as a consequence, the sensitivity of these homozygous deletion of the candidate tumor suppressor genepl6 (MTSJ/ cells to certain therapeutic agents. Because deletion of these innocent CDKN2), which resides in the chromosome 9p21 region. Here we: (a) examined a seties of these cell lines for the Incidence ofcodeletion of genes bystander genes occurs in cells undergoing oncogenic transformation located near p16, in particular, the gene for the enzyme 5'-deoxy-S'- but not in normal cells, it may be possible to design therapeutic methylthioadenosine phosphorylase (MTAP) and the genes of the IFN.a strategies that take advantage of these selective genetic losses. and 43 cluster (IFNs); and (b) investigated whether therapeutic strategies A potential example of this involves malignant cells that have could be developed that target malignant cells that have undergone the undergone genetic deletions in the chromosome 9p2l region. The codeletion of such genes. Five of the eight pancreatic carcinoma cell lines genes for two candidate tumor suppressor proteins, pl6'@4 and were plt$@, MTAP was codeleted in all five cases. Because MTAP phos. pl5@4'@, respectively referred to aspl6 (MTSI/CDK4I/CDKN2) and phorolyzes 5'-deoxy-S'-methylthioadenosine (MTA), generated as a by p15(MTS2),residecloseto oneanotherin thisregion(2). Bothof product of polyamine synthesis, to the salvageable purine base adenine, these proteins act to inhibit the catalytic activity of the cyclin-depend loss of this pathway in p16W, @f7@4p cells might sensitize these cells to methotrexate (MTX), the mechanism of action of which involves, in part, ent kinase 4 (or 6)-cyclin D complex, which in its active form an Inhibition of purine de novo synthesis. MTAP@normal keratinocytes phosphorylates the retinoblastoma protein Rb (3, 4); this phosphoryl and pancreatic carcinoma lines had relatively poor sensitivity, In terms of ation is thought to be a key step in the progression of cells from a efficacy, to the purine nucleotide-starving actions of MTX. This may be in restriction point late in G1 into S-phase (5). Genetic deletion or part due to the MTAP-dependent salvage of adenine moieties from endo functional inactivation via mutation of one or both of these cyclin genously generated MTA, because the MTAP inhibitor 5'-chloro.S'-de dependent kinase inhibitors might, in principle, contribute to a loss of oxyformycin A potentiates the antipurine actions of MTX in some of these the cell's ability to control cell cycle progression, a hallmark of MTAP@ @jflesAlso, exogenous MTA (10 gEM)reverses the growth-inhibi malignancy (2, 6). Indeed, homozygous deletion or point mutations of tory actions of MTh in these lines. In contrast, MTAP cell lines, which cannot recycle purines from endogenous MTA, have a relatively high the gene for p16 have been shown to occur in a variety of primary sensitivity to the antipurine actions of MTX, which is not modulated by malignancies (7—18).Where studied, the p1.5 gene, which resides 5'-chioro-S'-deoxyformycin A or exogenous MTA. Thus the MTAP loss in approximately 20 kb centromencally from p16 (2, 12), has been malignant cells may be an example of gene deletion chemoselectivity, in shown to be codeleted frequently in malignant cells that bear homozy which genetic deletions that occur as part of the oncogenic process render gous deletions of pitS (8, 9, 12, 14, 15). It has been suggested that these cells more sensitive to particular anticancer agents than normal homozygous deletions within the 9p2l region may be relatively cells, which have not undergone such deletions. We also examined whether common in particular cancers, because such deletional events could the loss of IFN genes sensitize cells to the growth-inhibitory actions of inactivate pl6, plS, and possibly other tumor suppressor genes resid these cytokines. Three of the five pltS@cell lines bore homozygous dele ing in this region simultaneously, resulting in a selective growth tions of IFNAJ and IFNBJ genes, representing each end of the IFN-a41 gene duster; one cell line bore a codeletion ofthe IFNA1 gene but retained advantage (12). the IFNBJ locus. Whereas the cell lines that were most sensitive to the When such deletional events involving multiple loci take place growth-inhibitory effects 0fIFN.fi or WN-a2b tended to be those with IFN within the 9p2l region, a number of other neighboring genes can be deletions, there were enough exceptions to this pattern to indicate that the lost coincidentally. Examples of this include the gene that encodes the IFN genotype does not reliably predict IFN responsiveness. enzyme MTAP3 (MTAP), which is reported to reside less than 100 kb from pitS (19), and the IFN gene cluster, consisting of the WN-@3@ INTRODUCTION gene (IFNBJ) and at least 25 genes and pseudogenes for IFN-a (IFNA) and IFN-(&)(20), located approximately 500—1000kb in the Loss or inactivation of tumor suppressor genes occurs in many telomeric direction from p16 (19). MTAP is codeleted with a fre cancers (1). In some cases, both tumor suppressor gene alleles are lost quency of >85% in cell lines bearing pitS deletions, whereas the IFN by mechanisms involving genetic deletion, i.e. , homozygous deletion. gene cluster is deleted in whole or in part in >50% of p16-deficient As result of these events, genes that reside nearby but are otherwise cell lines (21). unrelated to these tumor suppressor genes can frequently be codeleted. Hypothetically, loss of either MTAP or the IFN genes might render malignant cells with 9p21 defects more sensitive to certain antineo Received 9/21/95; accepted 1/3/96. plastic agents than normal cells that have not undergone these genetic Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page charges. This article must therefore be hereby marked advertisement in accordance with deletions. MTAP phosphorolyzes the nucleoside MTA, a metabolite 18U.S.C.Section1734solelyto indicatethisfact. of S-adenosylmethionine produced during the synthesis of the poly I This work was supported by PHS Grant ROl CA-6378l (T. M. S.). The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the PHS. 3 The abbreviations used are: MTAP, 5'-deoxy-5'-methylthioadenosine phosphonyl 2 To whom requests for reprints should be addressed. at Cancer Center, Room HB-774, ase; 5'-CIF, 5'-chloro-5'-deoxyfonmycin A; LU, international unit; KGM, kenatinocyte University of Massachusetts Medical Center, 55 Lake Avenue North, Worcester, MA growth medium; MTA, 5'-deoxyS'-methylthioadenosine; MTX, methotrexate; MU, 3-(4,5- 01655. dimethylthiazol-2-yl)-2i-diphenyltetnazolium bromide; IC,,, 50% inhibitory concentration. 1083 p16. MTAP. AND 1FN DELETION AND CHEMOSELECTIVITY amines spermidine and spermine, to yield 5-methylthioribose-l-phos a solution of MiT (5 mg/mI dissolved in PBS) were added directly to each phate, a methionine precursor, and adenine (22), which is converted to well, and the plate was incubated an additional 2 h at 37°C. A solution of 0.1 adenine nucleotides via adenine phosphoribosyltransferase (EC N HC1 and 10% Triton X-lOO in isopropanol was added to each well (1.2 ml) 2.4.2.7; Refs. 23 and 24). Thus, MTAP plays a critical role in to dissolve the colored metabolite of MTI', formazan. This solubilization was recycling adenine moieties from S-adenosylmethionine, derived orig facilitated by sonicating each well using a Branson 250 sonifier (Danbury, CT). An aliquot (300 @l)of each well was then transferred to individual wells inally from AlP, back to adenine nucleotide pools (25). MTAP in a 96-well plate, and the absorbance at 570 nan was measured in a Molecular deficient malignant cells, unable to metabolize MTA, simply excrete Devices (Sunnyvale, CA) plate reader against a background wavelength of 650 it (26). Loss of this adenine salvage pathway might sensitize p16@, nm. After substraction of background values (wells containing medium but no MTAP cells to the anticancer agent MTX, the mechanism of action cells), the absonbance values were plotted as a percentage of the absorbance of which involves, in part, purine nucleotide starvation via an inhibi value of control wells, in which cells were grown in the absence of MTX.