Induction of the Common Fragile Site FRA3B Does Not A€Ect FHIT

Induction of the common fragile site FRA3B does not aect FHIT expression

Dagmar Michael*,1,2 and Manfred F Rajewsky2

1Department of Internal Medicine (Cancer Research) University of Essen Medical School and West German Cancer Center, Hufeland-Strasse 55, D-45122 Essen, Germany; 2Institute of Cell Biology (Cancer Research), University of Essen Medical School and West German Cancer Center, Hufeland-Strasse 55, D-45122 Essen, Germany

A long-standing issue concerns the extent to which are speci®c regions that appear as gaps and breaks in fragile sites predispose to cancer-associated chromosomal metaphase chromosomes when DNA replication is rearrangements. The FHIT gene at chromosome 3p14.2 perturbed (for review, see Sutherland et al., 1998). spans the most common fragile site, FRA3B, in the Interest in fragile sites has been further stimulated by human genome. Although the FHIT gene is altered in cloning of the fragile histidine triad (FHIT) gene (Ohta many human cancers, its status as a tumor suppressor et al., 1996) which spans the most common fragile site gene has remained controversial, particularly since in the human genome, FRA3B, at chromosome 3p14.2. functional studies provided contradictory results. It had This region shows loss of heterozygosity (LOH) in a been suggested the FHIT alterations result from FRA3B variety of cancers. Although the FHIT gene is altered induction promoted by the interference of carcinogens in many human cancers (Ohta et al., 1996; Sozzi et al., with DNA replication. Here we investigated the eect of 1996) its status as a tumor suppressor gene has FRA3B induction on FHIT expression. Common fragile remained controversial, particularly since functional sites were induced by treatment with aphidicolin and studies provided contradictory results (Siprashvili et scored cytogenetically. FHIT transcription was analysed al., 1997; Otterson et al., 1998; Ji et al., 1999; Wu et by RT ± PCR and RNase protection analysis. Unexpect- al., 2000). Moreover the signi®cance of the FHIT gene edly, FHIT transcription proceeded unchanged after in carcinogenesis has been questioned since Fhit null fragile site induction. Aberrant FHIT transcripts lacking mice (Fhit 7/7) were reported to be long-lived and one or more exons were not observed. Morevover, fertile (Fong et al., 2000). Western blots revealed that the levels of FHIT prior to It has previously been proposed that FHIT altera- and following fragile site induction was unchanged, tions found in tumors result from the induction of whereas p53 was found at elevated levels after induction. FRA3B, possibly promoted by the interference of FRA3B induction thus has no direct eect on FHIT carcinogens with DNA replication (Sozzi et al., 1996; transcription and translation. Oncogene (2001) 20, Fong et al., 2000). To examine this potential 1798 ± 1801. mechanism of mutagenesis, we have investigated the eect of fragile site induction on FHIT expression. Keywords: FHIT gene; FRA3B; fragile sites; human Common fragile sites were induced by treatment of chromosome 3 PHA-stimulated lymphocytes with aphidicolin (Le Beau et al., 1998), a speci®c inhibitor of DNA polymerases a, d and e. Heparinized blood samples were obtained from eight healthy donors, and three Examination of the relationships between DNA types of PHA-stimulated cultures were studied: damage and mutagenesis provides insights into the untreated control cultures, cultures treated with molecular mechanisms underlying cancer development. aphidicolin at a ®nal concentration of 0.2 mM,and The identi®cation and characterization of genomic cultures treated with aphidicolin at a ®nal concentra- regions that preferentially undergo DNA damage- tion of 0.4 mM. Controls using primary non-cultured induced rearrangements are important issues. In this normal peripheral blood lymphocytes from each donor respect, a long-standing question concerns the extent to were included. PHA-stimulated lymphocytes are most which fragile sites predispose to cancer-associated commonly used in studies of fragile sites, and in chromosomal rearrangements (Le Beau and Rowley, addition to aphidicolin various mutagens and carcino- 1984; Yunis and Soreng, 1984). Common fragile sites gens have been shown to induce chromosomal breaks at fragile sites in this experimental system (Yunis and Soreng, 1987). To ascertain the induction of fragile sites by exposure to aphidicolin, GTG-banded chro- *Correspondence: D Michael, Institute of Cell Biology (Cancer mosome preparations were analysed by standard Research), University of Essen Medical School and West German cytogenetic techniques (Barch, 1991) (data not shown). Cancer Center, Hufeland-Strasse 55, D-45122 Essen, Germany Received 28 September 2000; revised 4 January 2001; accepted 9 Initially FHIT transcription was analysed prior to January 2001 and following fragile site induction. Transcripts com- FRA3B fragile site and FHIT expression D Michael and MF Rajewsky 1799 prising the entire open reading frame were ampli®ed by clease (RNase) protection analysis was performed as RT ± PCR using primers corresponding to positions 261 described (Gilman, 1993), using an antisense probe (exon 4) and 1080 (exon 10) of the human FHIT cDNA covering the FHIT mRNA from nt 148 to nt 833. As sequence (Ohta et al., 1996; GenBank accession number shown in Figure 1b, FHIT mRNA levels were almost U46922). To avoid artefacts during nested ampli®ca- unaected by treatment with aphidicolin (lanes 3 ± 6). tion, a robust single-stage RT ± PCR ampli®cation Control experiments using increasing amounts of protocol was applied (Thiagalingam et al., 1996). Upon mRNA showed that the intensity of the protected treatment with aphidicolin (0.2 and 0.4 mM) only the bands correlated with amounts of mRNA, con®rming normal-sized 820 nt FHIT transcript was detected in the that the antisense probe was in high molar excess (data samples from all eight donors (Figure 1a). This not shown). transcript was shown to be identical to the FHIT wild- It has been reported that FHIT translation may be type transcript by sequence analysis (data not shown). repressed despite ecient transcription (Huebner et al., Aberrant FHIT transcripts lacking one or several exons 1998). Therefore FHIT translation was analysed as were not observed. Since aberrant transcripts lacking well. Consistent with the results of RT ± PCR and exons 3 or 4 have been reported (Ohta et al., 1996; Sozzi RNase protection experiments, Western blots revealed et al., 1996) a primer for exon 1 (position 140 of the the same high level of FHIT protein prior to and FHIT cDNA) was used in combination with the exon 10 following fragile site induction (Figure 2, upper panel). primer to amplify nearly the entire FHIT mRNA. Thus, neither FHIT transcription nor translation were However, again only the expected normal-sized 941 nt signi®cantly altered by the induction of fragile sites. FHIT transcript was detected. Upon their induction, fragile sites are hot spots for Although RT ± PCR is a sensitive method, minor sister chromatid exchange (SCE) (Glover and Stein, eects, e.g. on the rate of transcription, may have gone 1987; Wenger et al., 1987). As a key step SCE involves undetected due to the semiquantitative nature of the generation of DNA double-strand breaks, i.e., a standard PCR assays. Therefore quantitative ribonu- type of DNA lesion capable of triggering p53-

Figure 1 Fragile site induction does not interfere with FHIT transcription. (a) Southern blot analysis of the RT ± PCR products from RNA of normal lymphocytes (1) and PHA-stimulated lymphocytes, either untreated (2) or exposed to aphidicolin at concentrations of 0.2 mM (3) or 0.4 mM (4). For hybridization a FHIT-speci®c 32P-labeled cDNA probe comprising nt 261 to 1080 was used. Lanes marked + and 7 correspond to RT-reactions with and without reverse transcriptase. Migration of molecular weight markers is indicated in bp on the left margin. (b) RNase protection assay from mRNA of normal lymphocytes (3) and PHA- stimulated lymphocytes, either untreated (4) or exposed to aphidicolin at concentrations of 0.2 mM (5) or 0.4 mM (6). Four mgof poly(A)+ were bybridized with 56105 c.p.m. of gel puri®ed 32P-labelled antisense probe in 30 ml formamide buer. After RNase A/ RNase T1 and subsequent proteinase K digestion, the samples were phenol-extracted and loaded onto a 6% sequencing gel. Lanes marked (1) and (2) show the undigested probe (1) and a control reaction with yeast tRNA (2). The fragment size of the pBluescript HpaII molecular weight marker is indicated in bp on the left margin and, therefore, does not precisely re¯ect the size of the unprotected and protected RNA fragments

Oncogene FRA3B fragile site and FHIT expression D Michael and MF Rajewsky 1800 translationally, p53 was examined by Western blot analysis (Figure 2, central panel). Whereas in normal lymphocytes p53 was hardly detectable (lane 1), p53 accumulation was readily detected in PHA-stimulated lymphocytes (lane 2). This is in agreement with previous results demonstrating upregulation of p53 after triggering resting cells into proliferation (Mosner and Deppert, 1994; Mummenbrauer et al., 1996). p53 further accumulated upon exposure to 0.2 mM aphidicolin, and an even higher level was reached upon exposure to 0.4 mM aphidicolin (lanes 3 and 4), indicating DNA damage-induced p53 activity. In summary, the present analyses demonstrate that FRA3B induction has no direct eect on FHIT transcription and translation. Since the FRA3B region is late replicating, and exposure to aphidicolin further delays replication, thus resulting in incompletely replicated DNA (Le Beau et al., 1998; Wang et al., 1999), FHIT transcription may be completed before the start of replication. Alternatively, the present results could be explained by an intact p53 pathway and/or repair of DNA double-strand breaks by homologous recombination. The alterations of the FHIT gene frequently observed in tumors are thus more likely to Figure 2 Fragile site induction does not interfere with FHIT occur in cancer cells harboring p53 mutations and/or translation and elevates p53 protein levels. Whole cell extracts defects in recombinational repair. Interestingly, a (20 mg/lane) from normal lymphocytes (1) and PHA-stimulated correlation between FHIT deletions in tumors and lymphocytes, either untreated (2) or exposed to aphidicolin at concentrations of 0.2 mM (3) or 0.4 mM (4), were analysed by their p53 mutation status has been reported (Boldog et Western blotting with a rabbit anti-FHIT antiserum (upper panel) al., 1997). Whether or not FHIT inactivation may and the mouse monoclonal antibody DO-1 speci®c for human p53 contribute to the phenotype of cancer cells thus still (central panel). Equivalent protein concentrations (20 mg/lane) in remains an open question. extracts were con®rmed by separate analysis of actin (lower panel)

Acknowledgments dependent DNA damage response pathways (Nelson Supported by the IFORES Program of the University of and Kastan, 1994; Huang et al., 1996). We therefore Essen Medical School. We thank Dr Andrea Kindler- investigated whether the p53 pathway was activated in RoÈ hrborn for discussions and review of this manuscript the experimental system used here. Since the amount and Dr C Kirch for providing the anti-p53 monoclonal and the activity of p53 are mainly regulated post- antibody.

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