Expression of truncated FHIT transcripts in cervical cancers ... - Nature

Oncogene (2001) 20, 4665 ± 4675 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc

Expression of truncated FHIT transcripts in cervical cancers and in normal human cells CP Matthews*,1,2, K Shera1, N Kiviat2 and JK McDougall1,2 1

Fred Hutchinson Cancer Research Center, Cancer Biology Program, 1100 Fairview Avenue N., P.O. Box 19024, Seattle, Washington, WA 98109-1024, USA; 2Department of Pathology, University of Washington, Box 357470, 1959 Paci®c Street N.E., Seattle, Washington, WA 98195-7407, USA

To analyse FHIT transcription patterns in cervical cancer, a series of primary cervical tumors and normal control samples were studied using RT ± PCR. Full length and truncated FHIT transcripts were detectable in all samples tested. Interestingly, the expression of truncated FHIT transcripts by primary epithelial cells in vitro was associated with con¯uency. The breakpoints of most transcript deletions coincided with genuine splice site sequences, suggesting that they resulted from alternative splicing. These ®ndings demonstrate that truncated FHIT transcripts are commonly detected in both normal and tumor tissues, and suggest that these altered transcripts are not causally related to tumorigenesis in cervical cancer. Oncogene (2001) 20, 4665 ± 4675. Keywords: cervical carcinoma; FHIT; alternative transcripts Introduction Frequent losses of heterozygosity (LOH) on the short arm of chromosome 3 (3p) have been identi®ed in cervical cancer and its precursor lesions (Yokota et al., 1989; Kohno et al., 1993; Mitra et al., 1994; Larson et al., 1997; Wistuba et al., 1997; Chu et al., 1998; Rader et al., 1998). One commonly deleted region is 3p14 (Kohno et al., 1993; Larson et al., 1997; Wistuba et al., 1997; Chu et al., 1998; Rader et al., 1998; Matthews et al., 2000). A candidate tumor suppressor gene named FHIT (fragile histidine triad) has been cloned and mapped to 3p14.2 (Ohta et al., 1996). FHIT is a large gene, composed of 10 exons that span more than 1 megabase of genomic DNA. The locus includes the most active fragile site in the human genome, FRA3B, which spans introns 3 through 5 of FHIT (Ohta et al., 1996, Zimonjic et al., 1997). The coding region begins in exon 5 and ends in exon 9 and is predicted to encode a 147-amino acid protein with a mass of approximately 16.8 kD. Fhit protein belongs to the histidine triad superfamily (HIT) of nucleotide binding proteins and

*Correspondence: CP Matthews; E-mail: [email protected]. Received 20 November 2000; revised 26 April 2001; accepted 8 May 2001

has been shown to function as a diadenosine triphosphate (Ap3A) hydrolase (Barnes et al., 1996) and interact with tubulin (Chaudhuri et al., 1999) in vitro. Several published reviews have summarized observations supporting FHIT's possible role as a tumor suppressor gene targeted by 3p14 LOH in cervical and several other types of cancer (Druck et al., 1998; Huebner et al., 1998; Le Beau et al., 1998; Mao, 1998). For example, the breakpoint of the t(3;8)(p14.2;q24) translocation, which occurs in a kindred with familial renal cell carcinoma, maps within the FHIT locus. Homozygous deletions within the FHIT gene, some of which include coding exons, have also been identi®ed in several primary tumors and cell lines. Moreover, alternate FHIT transcripts have been detected in many of the tumor types that exhibit frequent deletions of the 3p14.2 region. A role for Fhit in the regulation of apoptosis and the cell cycle has been proposed in recent studies (Ji et al., 1999; Sard et al., 1999). Fhit overexpression has been reported to suppress tumorigenicity of Fhit-negative lung, gastric and renal cell carcinoma-derived cell lines (Siprashvili et al., 1997; Ji et al., 1999). While these ®ndings support FHIT's possible role as a tumor suppressor, other reports question this view. It has been suggested that the frequent structural alterations of the FHIT locus simply re¯ect genetic instability of FRA3B. Detection of alternative transcripts does not always correlate with genetic alterations of the FHIT/FRA3B locus. Moreover, alternative FHIT transcripts of the types detected in cancers have been observed in histologically normal tissues (Boldog et al., 1997; Gayther et al., 1997; Panagopoulos et al., 1997; van den Berg et al., 1997), some homozygous deletions within FHIT aect only introns, and point mutations within the FHIT coding region are extremely rare (Boldog et al., 1997; Druck et al., 1997). A closer examination of the t(3;8)(p14.2;q24) translocation breakpoint in familial renal carcinoma revealed that the FHIT coding region is intact and FHIT is expressed. However, the open reading frame of a gene located on chromosome 8, TRC8, is interrupted (Gemmill et al., 1998). Functional studies of the Fhit protein's ability to suppress the tumorigenic phenotype of cancer cells have also yielded con¯icting results. In contrast to the analyses of lung, gastric and renal cell carcinoma-derived cell lines described above, Fhit

Expression of truncated FHIT transcripts CP Matthews et al

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protein expression failed to suppress tumorigenicity in Fhit-negative cervical carcinoma-derived cell lines (Otterson et al., 1998; Wu et al., 2000). A study of gene expression in cervical tumors demonstrated a twofold reduction of FHIT mRNA relative to normal cervical controls (Matthews, 2000). Here we further explore the role of the FHIT gene in the development of cervical carcinoma by RT ± PCR analysis of FHIT transcripts in primary cervical tumors and normal controls.

Results FHIT transcription in cervical squamous cell carcinoma To analyse FHIT transcription in cervical carcinoma, we performed nested RT ± PCR analysis of cDNA templates prepared from a series of primary cervical squamous cell carcinomas, normal cervical epithelium, and primary human foreskin keratinocytes (HFK's). PCR reactions were performed in duplicate using single oligo-dT primed cDNA preparations as template. Sample integrity was assessed by visual inspection of RNA on denaturing gels (Figure 1a) and b-actin PCR of cDNA preparations (Figure 1b). Normal, full-length FHIT transcripts (707 bp), together with FHITb, a normal splice variant (696 bp) (Mao et al., 1996), were isolated from cervical tumors, normal cervical epithelium, and primary HFK's (Figure 2). In addition, shorter, variable-length

RT ± PCR products were ampli®ed from both cervical tumors and controls. These truncated transcripts were not consistently ampli®ed in duplicate PCR ampli®cations of single cDNA preparations (Figure 2), suggesting that they represented transcripts expressed at very low levels, or alternatively, PCR artifacts. To investigate the origin of these truncated transcripts, the RT ± PCR products were cloned and sequenced, and the observed deletion breakpoints were correlated with the positions of known FHIT splice sites. A summary of FHIT transcripts isolated from cervical tumors and controls is presented in Figure 3. The deletion breakpoints coincided with known splice sites in 73% (22/30) of the RT ± PCR products isolated. Thus the vast majority of these truncated transcripts likely represent genuine FHIT splice variants. Alternate FHIT transcripts carrying a deletion of exon 8, alone or in combination with the FHITb deletion, were observed in all samples. The majority of alternative transcripts lacked exon 5 which contains the start codon for Fhit

A

B

Figure 1 Total RNA and b-actin cDNA from primary cervical tumors and normal controls. The quality of the total RNA isolated from primary cervical tumors (E229, E230, E233, E253), histologically normal cervix (Normal), and primary HFK's (HFKp4 and HFKp6) was assessed by visual inspection of ethidium bromide-stained denaturing gels (a). 0.24 ± 9.5 Kb RNA ladder (Life Technologies) was used as marker on the denaturing gels. The integrity of all cDNA samples was veri®ed by successful ampli®cation of b-actin message (b). The ampli®ed band corresponding to b-actin message is 510 bp and is indicated by an arrow. One ml of ddH2O was used as template in the negative control reaction. 100 base pair ladder (Amersham Pharmacia Biotech) was used as size marker Oncogene

Figure 2 Ampli®cation by nested-PCR of FHIT cDNA from primary cervical carcinomas and normal control samples. Primary cervical tumors (E229, E230, E233, E253), histologically normal cervix (Normal), and primary HFK's (HFKp4 and HFKp6) were analysed. The cDNA was ampli®ed with primers 5U2 and 3D2 followed by nested primers 5U1 and 3D1. All PCR reactions were performed in duplicate (a and b). The ampli®ed band corresponding to a normal message is 707 bp and is indicated by an arrow. Smaller bands represent alternative FHIT transcripts. 100 base pair ladder (Amersham Pharmacia Biotech) was used as size marker (M). One ml of ddH2O was used as template in the negative control (N)


protein translation, and are likely not translated in vivo. Finally, some transcripts isolated from both cervical tumors and normal keratinocytes retained short segments of FHIT intron 5 sequence. Interest-

A

ingly, FRA3B spans introns 3 through 5 of the FHIT gene. The detection of truncated FHIT transcripts in both cervical tumors and normal cervical cells does not support a speci®c involvement of these alternative

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ORF All All E230 E253 E229, E230, E253 E233, E253 E253 E229 E230 E230 E233 E233 E233 E233 E233 E233 E233 5U1/3D1 primer sequences genomic sequence from FRA3B/FHIT locus * alternative splicing of E9/E10 boundary

B

ORF All All HFKp4 HFKp4 HFKp6 HFKp6 HFKp6 HFKp6 HFKp6 HFKp6 HFKp6 Normal Cx Normal Cx Normal Cx 5U1/3D1 primer sequences genomic sequence from FRA3B/FHIT locus * alternative splicing of E9/E10 boundary

Figure 3 A schematic presentation of the FHIT messages detected in primary cervical carcinomas, normal cervix and primary HFKs. FHIT transcripts detected in tumors (E229, E230, E233, E253) are summarized in (a). The transcripts from normal controls (Normal cervix, HFK p4, HFK p6) are shown in (b). Primers 5U1 and 3D1 amplify nucleotides 201 to 907. The open reading frame (ORF) begins at nucleotide 363 and ends at nucleotide 806. The blue boxes indicate primer positions. Exon numbers are shown within the boxes. A thin black line indicates deletion. Green boxes represent insertion of genomic material from the FRA3B/FHIT locus. An asterisk symbolizes alternative splicing of the exon 9/10 boundary Oncogene


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A

B

Figure 4 Ampli®cation of FHIT cDNA from cultured cells. Primary epithelial cells (HFK), immortalized epithelial cells (cl 22 and cl 398) and primary ®broblasts (HFF) were analysed. RNA was isolated from each cell type at four time points, 50% con¯uency (1), con¯uent for 24 h (2), con¯uent for 72 h (3), and after release from con¯uence (4). The ®broblasts were also harvested after 24 (5) or 72 h (6) of serum starvation and within 48 h after return of serum (7). The cDNA was ampli®ed with primers 5U2 and 3D2 followed by nested primers 5U1 and 3D1. All FHIT PCR reactions were performed in duplicate (a and b). Three ml of each PCR product was analysed on 2% 3 : 1 Nusieve agarose gel. The ampli®ed product corresponding to a normal message is 707 bp (indicated by the arrow). The smaller bands correspond to alternatively spliced and FHIT transcripts. Note the absence of smaller transcripts in primary HFK's that are subcon¯uent (lanes 1 and 4). One ml of ddH2O was used as template in the negative control reaction (N) 100 base pair ladder (Amersham Pharmacia Biotech) was used as size marker (M) Oncogene


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transcripts in cervical tumorigenesis. Furthermore, one might question the physiological signi®cance of transcripts expressed at such low levels. FHIT transcription in cultured normal cells A recent study of FHIT expression patterns in primary lymphocytes indicates that these abnormal transcripts may represent reduced RNA-splicing ®delity in aging cells. According to Gayther et al. (1997), normal primary lymphocytes analysed immediately after blood draw did not express alternative FHIT transcripts by RT ± PCR. However, the same isolate of cells, after `aging' by incubation at room temperature for 60 h expressed truncated FHIT transcripts similar to those detected in many cancers. To investigate the eect of con¯uency on FHIT expression, we puri®ed RNA from primary human foreskin ®broblasts (HFF's), primary HFK's, and hTERT-immortalized epithelial cell lines at several degrees of con¯uency. Cells were harvested for RNA extraction at four time points, 50% con¯uency, 24 h at 100% con¯uency, 72 h at 100% con¯uency, and within 48 h after release from con¯uency. HFF's were also harvested after 24 or 72 h of serum starvation and within 48 h after return of serum. Oligo(dT) primed cDNA synthesis was then performed. All of the cDNA samples were screened simultaneously by nested-PCR for deletions in FHIT transcripts. Truncated FHIT transcripts were absent in three independent isolates of sub-con¯uent primary HFK's (Figure 4a, lane 1). Interestingly, abnormal transcripts were detectable after these cells reached con¯uency (Figure 4a, lanes 2 and 3), but decreased after the cells were released from con¯uency (Figure 4a, lane 4). One hTERT-immortalized epithelial cell line, cl 398, exhibited a FHIT transcription pattern similar to that seen in primary epithelial cells (Figure 4a, lanes 1 ± 4), although another hTERT-immortalized epithelial cell line, cl 22, did not (Figure 4a, lanes 1 ± 4). Primary HFF's failed to show an association between con¯uency (Figure 4b, lanes 1 ± 4), or serum starvation (Figure 4b, lanes 1, 5 ± 7), and expression of truncated FHIT transcripts. In these cells, abnormal transcripts were detectable at low level in con¯uent (Figure 4b, lanes 2 and 3) and sub-con¯uent cultures (Figure 4b, lanes 1 and 4), as well as in serum starved (Figure 4b, lanes 5 ± 7) and serum supplemented (Figure 4b, lanes 1 ± 4) cultures. Sample quality was con®rmed by visual inspection of RNA on denaturing gels (Figure 5a) and b-actin PCR analysis of cDNA preparations (Figure 5b). The detection of truncated FHIT transcripts in con¯uent but not in sub-con¯uent normal HFK's suggests an association between alternative transcripts and cell cycle arrest in keratinocytes. In the hTERT-immortalized cl 22 cells, however, alternative transcripts were detected in sub-con¯uent cultures, indicating that some immortalized epithelial cell lines might express detectable levels of truncated transcripts in the absence of arrest.

Figure 5 Total RNA and b-actin cDNA from cultured primary HFK's. Total RNA was isolated from primary HFK's at four time points, 50% con¯uency (1), con¯uent for 24 h (2), con¯uent for 72 h (3), and after release from con¯uence (4). The integrity of the RNA was assessed by visual inspection of ethidium bromidestained denaturing gels (a). 0.24 ± 9.5 Kb RNA ladder (Life Technologies) was used as marker on the denaturing gels. The quality of all cDNA samples was veri®ed by successful ampli®cation of b-actin message (b). The expected PCR product is 510 bp in length and is indicated by an arrow. One ml of ddH2O was used as template in the negative control reaction (N) 100 base pair ladder (Amersham Pharmacia Biotech) was used as size marker (M)

To investigate whether cell cycle arrest correlated with alternative transcript expression, FACS analysis was performed in parallel with RT ± PCR. As expected, both the primary HFK's and hTERT immortalized cell cultures arrested in G1 after 3 days of complete con¯uency (Figures 6 and 7). This result supports an hypothesis that expression of alternative FHIT transcripts is associated with cell cycle arrest in primary keratinocytes. To determine if expression of alternative FHIT transcripts was associated with genomic rearrangements within the FHIT locus, RFLP analysis was performed. We did not detect genomic rearrangements in any of the primary cells or immortalized cell lines by Southern blotting using a FHIT cDNA probe spanning exons 3 ± 10 (data not shown). Therefore, expression of alternative transcripts in con¯uent epithelial cells does not indicate genomic rearrangement of the FHIT locus. Expression patterns of other transcripts To assess whether the dierences observed in transcripts generated by sub-con¯uent and con¯uent cells re¯ected a global transcriptional error or one speci®c to FHIT, we analysed the same series of samples for the presence of truncated transcripts in other genes. Because `age'-associated alternative transcripts have previously been reported for TSG101 (Gayther et al., 1997), fragments spanning multiple exons were ampli®ed for TSG101. TSG101, a candidate tumor suppressor gene that maps to 11p15 (Li et al., 1997), is in close Oncogene


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intron/exon boundaries in the coding and 5' untranslated regions of the gene, are similar to those detected in FHIT. A third gene, HPRT1, which is not located near a fragile site, was also analysed. There was no evidence of alternative transcripts in HPRT1 (Figure 8), however, truncated transcripts of TSG101 were detected (Figure 9). Nested-PCR analysis of subcon¯uent and con¯uent cell cultures identi®ed TSG101 transcripts shorter than full-length in all cases tested. These ®ndings demonstrate that expression of con¯uency associated alternative transcripts is not a global phenomenon. Discussion Figure 6 Cell cycle pro®le of primary HFK's. The pro®les presented here are those of a single isolate of primary HFK's (HFK III) harvested at four levels of con¯uency, 50% (a), 100% for 1 day (b), 100% for 3 days (c), and 2 days after release from con¯uency (d). The DNA content of the cells was estimated based on the intensity of PI staining measured by FACS. A second isolate of HFK's also generated a similar pro®le. G1 arrest would be expected after 3 days at 100% con¯uency (C)

Figure 7 Cell cycle pro®le of cl 22 cells. hTERT immortalized cl 22 were harvested at four time points, 50% con¯uency (a), 100% con¯uency for 1 day (b), 100% con¯uency for 3 days (c), and 1 day after release from con¯uency (d). As described in the previous ®gure, PI-staining intensity was used to estimate the number of cells in each phase of the cell cycle. As expected, cell cycle progression was arrested in G1 by long-term con¯uency (c)

proximity to the aphidicolin inducible fragile site, FRA11C. TSG101 has been described as altered in a variety of cancers, including breast, ovarian, lung, and prostate (Gayther et al., 1997; Li et al., 1997; and Sun et al., 1997). The mutations reported for TSG101, large deletions within transcripts with breakpoints at the Oncogene

Several groups have reported the presence of tumorrelated alternative transcripts of FHIT in many types of primary tumors and tumor-derived cell lines. However, others have detected similar alternative transcripts in normal control tissues and cell lines (Le Beau et al., 1998; Wang and Chang, 1999). Our demonstration of altered FHIT transcripts in both primary cervical tumors and normal controls does not support a speci®c involvement of these alternative transcripts in tumorigenesis. In all of the cases we examined, normal-sized FHIT transcripts were expressed at robust levels. However, faint bands of smaller size were also present in all samples tested. Sequence analysis revealed that these smaller bands represented alternatively spliced transcripts of FHIT. FHITb, a normally occurring alternatively spliced form of FHIT missing 11 nt at the beginning of exon 10, was detected in all samples. The signi®cance of this 11 nt deletion is unknown. Recently, it has been reported that transcripts with deletions of all upstream non-coding exons and deletion of the 11 bp at the beginning of exon 10 cannot be translated in vitro, while transcripts missing only the upstream non-coding sequences can be translated into protein (Ferrer et al., 1999). Transcripts missing exon 8 were found in all samples analysed. This type of transcript was identi®ed as an aberration in head and neck carcinomas (Virgilio et al., 1996), and as an alternatively spliced form of FHIT in normal fetal brain (Boldog et al., 1997). Exon 8 contains the conserved histidine triad motif and a possible zinc binding site (Lima et al., 1996). Removal of this exon would inactive the Ap3A hydrolase activity of the protein. Transcripts with various deletions of exons 5 ± 9 into which genomic sequences had been inserted were identi®ed in primary HFKs and an invasive SCC. Similar alternative transcripts were recently reported to be cancer-speci®c in a series of cervical carcinomas (Segawa et al., 1999). However, detection of these transcripts in primary HFKs, reported here, does not support the view that they are cancer-speci®c. Various deletions of exons 4 ± 9 were also detected in both normal controls and tumor samples. The detection of similar alternative transcripts in both normal control


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Figure 8 Nested PCR analysis of HPRT1 cDNA. Primary epithelial cells (HFK), immortalized epithelial cells (cl 22 and cl 398) and primary ®broblasts (HFF) were analysed. RNA was isolated from each cell type at four time points, 50% con¯uency (1), con¯uent for 24 h (2), con¯uent for 72 h (3), and after release from con¯uence (4). The ®broblasts were also harvested after 24 (5) or 72 h (6) of serum starvation and within 48 h after return of serum (7). cDNA was ampli®ed with H1 and H2 primers followed by the nested primers H3 and H4. Three ml of each PCR product was analysed by electrophoresis on 2% 3 : 1 agarose. The ampli®ed product corresponding to normal HPRT1 transcripts (identi®ed by the arrow) is 561 bp in length and spans exons 3 through 9 of the cDNA. One ml of ddH2O was used as template in the negative control reaction (N) 100 base pair ladder (Amersham Pharmacia Biotech) was used as size marker (M)

Figure 9 Ampli®cation by nested PCR of TSG101-cDNA. Primary epithelial cells (HFK), immortalized epithelial cells (cl 22 and cl 398) and primary ®broblasts (HFF) were analysed. RNA was isolated from each cell type at four time points, 50% con¯uency (1), con¯uent for 24 h (2), con¯uent for 72 h (3), and after release from con¯uence (4). The ®broblasts were also harvested after 24 (5) or 72 h (6) of serum starvation and within 48 h after return of serum (7). The oligo(dT)-primed cDNA was ampli®ed by a series of nested primers, P1 and P2 followed by P3 and P4, speci®c for TSG101. All PCR reactions were performed in duplicate (a and b) and are presented above. Six ml of each sample was analysed by gel electrophoresis. The expected PCR product generated by the P3/ P4 primer set is 1145 bp in length (indicated by the arrow). Smaller products, corresponding to alternatively spliced transcripts, were detected in all samples tested. One ml of ddH2O was used as template in the negative control reaction (N) 100 base pair ladder (Amersham Pharmacia Biotech) was used as size marker (M) Oncogene


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Table 1 Nested and control PCR primers FHIT Primer

Sequence

Length

%GC

Tm (8C)

Position

Reference sequence

5'-ATCCTGGAAGCTTTGAAGCTCA-3' 5'-TCACTGGTTGAAGAATACAGGA-3' 5'-TCCGTAGTGCTATCTACATC-3' 5'-CATGCTGATTCAGTTCCTCTTGC-3'

22 22 20 23

45 41 45 48

53 51 50 55

141-162 954-933 201-220 907-885

U46922 U46922 U46922 U46922

TSG101 Primer P1 P2 P3 P4

5'-GCGGTGTCGGAGAGCCAGCTCAAGAAA-3' 5'-CCTCCAGCTGGTATCAGAGAAGTCAG-3' 5'-AGCCAGCTCAAGAAAATGGTGTCCAAG-3' 5'-TCACTGAGACCGGCAGTCTTTCTTGCTT-3'

27 26 27 28

59 54 48 50

64 61 60 61

94-120 1285-1260 106-132 1253-1226

NM006292 NM006292 NM006292 NM006292

HPRT1 Primer H1 H2 H3 H4

5'-TTATGCTGAGGATTTGGAAAGG-3' 5'-AAAAGCTCTACTAAGCAGATGG-3' 5'-TGTGATGAAGGAGATGGGAG-3' 5'-TTTTACTGGCGATGTCAATAGG-3'

22 22 20 22

41 41 50 41

51 51 52 51

166-187 846-825 241-260 801-780

M13642 M13642 M13642 M13642

b-actin Primer 5' b 3' b

5'-GAAATCGTGCGTGACATTAAG-3' 5'-CTAGAAGCATTTGCGGTGGAC-3'

21 21

43 52

50 54

660-680 1169-1149

X00351 X00351

5U2 3D2 5U1 3D1

and cervical tumor samples demonstrates that the alternative transcripts of the FHIT gene are not causally related to tumorigenesis in cervical cancer. Although there have been many reports of the expression of truncated FHIT transcripts in a variety of normal tissues (Gayther et al., 1997; van den Berg et al., 1997), only one previous study has focused on FHIT transcription patterns of primary cells in vitro (Gayther et al., 1997), the results indicating that alternative FHIT transcripts become more predominant as primary lymphocytes age. Our examination of transcription patterns of primary human keratinocytes and an hTERT-immortalized epithelial cell line indicated that expression of truncated FHIT transcripts was related to con¯uency of the cells in culture. However, there was no correlation between con¯uency and expression of truncated FHIT transcripts in the ®broblasts and or in a second immortalized line. The FHIT sequencing data presented above showed that the truncated transcripts result from alternative splicing. Analysis of TSG101 and HPRT1 transcripts was also performed to establish whether expression of truncated transcripts was speci®c for FHIT or a global occurrence. TSG101 exhibited many truncated transcripts. However, expression of these alternative TSG101 transcripts did not correlate with con¯uency in any of the cell lines tested in this study. HPRT1 did not exhibit alternative transcripts at any time point in any of the cell lines analysed, indicating that the expression of truncated transcripts was not a generalized phenomenon. Why do FHIT and TSG101 exhibit many alternative transcripts while HPRT1 displays none? The speci®city of exon joining is conferred by consensus sequences, known as canonical sequences, present at the exonintron boundaries of RNA transcripts (Reed and Maniatis, 1986). More recent studies indicate that sequence elements called exonic splicing enhancers Oncogene

(ESEs) also play an important role in directing alternative splicing (Tanaka et al., 1994; Ramchatesingh et al., 1995; Cooper and Mattox, 1997; Coulter et al., 1997). ESEs are purine rich sequences that direct the speci®c recognition of splice sites during constitutive and alternative splicing. An ESE-like sequence has been found within exon 3 of FHIT (Wang and Chang, 1999). Alternative splicing controlled by this ESE-like sequence might explain the deletion of downstream exons in FHIT transcripts. TSG101 is also thought to contain an ESE (Wang and Chang, 1999). HPRT1, on the other hand, has not been reported to contain an ESE-like sequence. It is also interesting to note that both FHIT and TSG101 map at or near a fragile site while HPRT1 does not. Expression of truncated transcripts by FHIT and TSG101 may simply re¯ect the instability of sequences within these fragile sites. Although the RNA splicing process exhibits a great deal of ®delity, mistakes clearly occur at a low frequency. The truncated FHIT transcripts described above might re¯ect rare spliceosome errors. One explanation for the increased expression of truncated FHIT transcripts in con¯uent primary keratinocytes is that there is a relaxation of transcriptional ®delity in con¯uent epithelial cells. One mechanism that might result in a relaxation of transcriptional ®delity is down-regulation of nonsense mediated RNA decay. The nonsense mediated RNA decay pathway degrades abnormally spliced mRNAs and prevents the production of truncated proteins. All eukaryotic cells possess an evolutionarily conserved mRNA degradation system that eliminates abnormal transcripts. The most highly conserved component of this mRNA surveillance system is the Upf1 protein (Applequist et al., 1997) and a dominant-negative mutant human UPF1 cDNA (hUPF1-R844C) has been described (Sun et al., 1998). Disruption of mRNA surveillance by expression of hUPF1-R844C protein in


primary human epithelial cells did not alter the expression of alternative FHIT transcripts (Matthews, 2000). Based on this observation, defective mRNA surveillance does not explain persistence of the alternative transcripts in con¯uent epithelial cells. The results of this study do not support a role for alternative FHIT transcripts in cervical cancer development. However, LOH of 3p14.2 is a common ®nding in cervical carcinoma as well as in other solid tumors. A continued search for other candidate tumor suppressor genes in this locus is justi®ed. Recently, expression of NRC-2, another putative tumor suppressor gene that maps to 3p14.2, has been reported to suppress tumorigenicity of renal carcinoma cell lines (Julicher et al., 1999). The role that NRC-2 plays in cervical cancer remains to be determined.

Materials and methods Tumor material

primer sets employed in each of these PCR reactions are presented in Table 1. Unless otherwise noted, the PCR reactions consisted of 1 ml template, 25 pmoles each primer, 200 mM each dNTP, 16PCR buer, 1.5 mM MgCl2, 2.5 units Platinum Taq antibody (Life Technologies), and 2.5 units Amplitaq (PE Biosystems). All ampli®cations were performed in a Perkin-Elmer 9600 thermocycler (PE Biosystems). Actual cycle times and temperatures are summarized in Table 2. All RT ± PCR products were separated on 2% 3 : 1 NuSieve (BioWhittaker Molecular Applications) agarose gels and visualized with 0.5 mg/ml ethidium bromide. b-actin RT ± PCR To verify the quality of the cDNA, a b-actin PCR was performed on each cDNA sample. The 5'- and 3'- b-actin primers were previously described by Segawa et al. (1999). In this case, Platinum Taq antibody was not added to the reaction mixture. All other reaction conditions were as described above. The expected b-actin PCR product is 510 bp in length. However, presence of a larger (717 bp) product would indicate genomic DNA contamination of the cDNA sample.

Cervical tumor specimens were collected as part of an NIH supported project (CA 75920). Histologic diagnosis was performed under the direction of the principal investigator, Dr Nancy Kiviat, at Harborview Hospital, Seattle. Of the four carcinomas, three were squamous cell carcinomas, and one was a carcinoma in situ (CIS). Normal cervical control tissue was obtained from patients who were treated for conditions other than cervical carcinoma at the University of Washington Medical Center, Seattle, Washington, USA. The tissues were frozen in liquid nitrogen and stored at 7808C.

The primers used for FHIT RT ± PCR analysis were ®rst described by Ohta et al. (1996). Primers 5U2 and 3D2 were employed in the initial ampli®cation. An aliquot of this ®rst PCR reaction was then used as template for the second PCR. In this second reaction, primers 5U1 and 3D1 amplify exons 3 through 10 of the FHIT cDNA. The reaction conditions were as described above, except the concentration of MgCl2 was increased to 2 ± 3 mM.

Cells and tissue culture

TSG101 RT ± PCR

Primary human foreskin keratinocytes (HFK's) and ®broblasts (HFF's) were cultured from neonatal foreskins. Clones 22 and 398 were picked by cylinder isolation from hTERT immortalized HFK's previously established by retroviral infection of HFK's with pLSXN vector containing the entire hTERT cDNA (Kiyono et al., 1998). All keratinocytes were grown in keratinocyte serum free media (KSFM) (Life Technologies) supplemented with bovine pituitary extract (Life Technologies) and epidermal growth factor (Life Technologies). The ®broblasts were cultured using Dulbecco's modi®ed essential medium (DMEM) supplemented with 10% FBS (Hyclone), penicillin (100 u/ml; Life Technologies) and streptomycin sulfate 100 mg/ml; Life Technologies).

Nested PCR of TSG101 transcripts was performed as described previously (Sun et al., 1997), with slight modi®cations. Brie¯y, primers P1 and P2 were used to amplify 1 ml of cDNA. One ml of this initial PCR product was then subjected to ampli®cation with the nested primers, P3 and P4.

RNA extraction and reverse transcription Total RNA was extracted using the commercially available RNeasy kit (Qiagen Inc.) and DNase treated with 1 unit RQ1 DNase (Promega) per mg RNA. All RNA samples were electrophoresed on 1.2% agarose, 2.2 M formaldehyde denaturing gels and visualized with 0.5 mg/ml ethidium bromide to con®rm sample integrity. First-strand cDNA was then synthesized using 50 mmoles oligo(dT), 100 units Superscript II RT (Life Technologies) and 1 mg DNase treated total RNA in a 20 ml reaction volume. Nested PCR analysis The expression patterns of four genes ± b-actin, FHIT, TSG101, and HPRT1 ± were examined by RT ± PCR. The

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FHIT RT ± PCR

HPRT1 RT ± PCR Nested PCR primers were designed with GCG version 10 (Genetics Computer Group) based on the complete HPRT1 cDNA sequence, GenBank accession number M31642. First an aliquot of cDNA was ampli®ed with the external primer set, H1 and H2. One ml of this ®rst PCR product was further ampli®ed with the internal primers, H3 and H4. Sequencing of FHIT transcripts RT ± PCR products derived from primers 5U1 and 3D1 were separated on 2% 3 : 1 Nusieve agarose (BioWhittaker Molecular Applications) gels. Normal-sized (707 bp) and truncated bands were cut out and eluted from the gel slices by using QIAquick spin columns (Qiagen Inc). The gelpuri®ed fragments were subcloned in vector pCR 2.1 using the TA cloning kit from Invitrogen. After blue-white selection, DNA mini-preparations of cloned PCR products were prepared and puri®ed with the Qiagen plasmid mini DNA isolation kit (Qiagen Inc). The puri®ed plasmids were sequenced by the Big Dye cycle sequencing method (PE Biosystems) using both forward and reverse M13 primers. Nucleotide sequences were analysed with the GCG version 10 Oncogene


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Table 2 RT ± PCR conditions Product

Denaturation Temp. Time

Annealing Temp. Time

Extension Temp. Time

b-Actin FHIT ext. FHIT int. TSG101 ext. TSG101 int. HPRT1 ext. HPRT1 int.

958C 958C 958C 958C 958C 958C 958C

558C 638C 638C 628C 628C 628C 628C

728C 728C 728C 728C 728C 728C 728C

30 30 30 30 30 30 30

s s s s s s s

30 30 30 40 30 30 30

s s s s s s s

90 90 90 90 90 90 90

s s s s s s s

Cycles 30 25 25 25 30 25 25

Note: All PCRs included an initial 2 min incubation at 958C and an additional 10 min 728C elongation cycle

(Genetics Computer Group) DNA analysis program. GenBank accession number U46922, the sequence of normal FHIT cDNA (Ohta et al., 1996), was the reference sequence for the analysis of alternative transcripts.

To determine the cell cycle pro®le of each culture, the DNA content of individual cells was estimated based on the intensity of PI staining. Southern blot analysis High molecular weight DNA was obtained from cultured cells using phenol-chloroform extraction (Sambrook et al., ed. 1989). Twelve mg of each sample was digested to completion with BamHI or HindIII. Digested DNA was separated by electrophoresis in 0.7% agarose gels and blotted onto Hybond N+ membranes (Amersham Pharmacia Biotech) by capillary transfer. The membranes were hybridized with a cDNA probe consisting of exons 3 ± 10 of the FHIT gene labeled with a32P-dCTP by random priming. Prehybridization and hybridization were carried out in 5% SDS, 0.5 M sodium phosphate, and 0.1 mg/ml salmon sperm DNA at 658C. Hybridized membranes were washed in 0.5% SDS, 50 mM sodium phosphate, ®rst at room temperature and then at 658C for 30 min each.

Cell cycle analysis Cells were harvested for cell cycle analysis at four time points: 50% con¯uency, 100% con¯uency, after 3 days at 100% con¯uency, and after release from con¯uency. FACS analysis was performed as described by White et al. (1990).

Acknowledgments We thank Jennifer Koop and Hadar Sheer for their excellent technical assistance, and the presented work was supported by NIH grant CA42792.

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