Inactivation of the Human Fragile Histidine Triad Gene at 3p14.2 in Monochromosomal Human/Mouse Microcell Hybrid-derived Severe Combined Immunodeficient Mouse Tumors¹

Overlapping HDs³have been found within the 3p21.3 region in three SCLC lines(1, 2, 3), in a BRC line, and a primary BRC(4). The minimum common deleted region was approximately 120 kb in length. (4). It included the LUCA1gene. In the 3p22–3p21.3 region adjacent to the DNA mismatch repair protein homologue gene, MLH1, HDs were detected in two lung tumor biopsies (5) and in five NSCLC lines, including ACC-LC5 (6, 7). The ITGA4L gene was cloned from the HD region in ACC-LC5 (8). HDs within 3p21.3 were also found in three lung tumors in vivo by FISH (9). A large 8-Mb deletion at the 3p13–3p12 was identified in one SCLC cell line, U2020 (10). A gene called Deleted in U-twenty twenty(DUTT1) was characterized as well (11).

The candidate tumor suppressor gene FHIT spans the FRA3B fragile site at 3p14.2 (12). In four CC, two GC, two nasopharyngeal, and one BRC carcinoma lines, the HDs at D3S1300(3p14.2) were shown to include parts of the FHIT gene(13). Loss of heterozygosity at 3p14.2 was very frequent in 32 lung cancer lines (100% of SCLC and 88% of NSCLC) and 108(45%) primary NSCLC (14). The same study identified HDs within the FHIT/FRA3B region in 6 (4.4%) of 135 lung cancer lines, whereas the Northern blot showed low or no FHITexpression in most of the lines (14). Hemi- or homozygous deletions, affecting exon 5 of FHIT preferentially, have been found in 86% of the early lesions classified as Barrett’s metaplasia and in 93% of the associated adenocarcinomas(15). Boldog et al. (16) found HDs within FHIT in nearly 90% of cervical and 50% of colorectal carcinoma cell lines. Recently, a HD within FHITwas found in a cervical carcinoma line (17). A HD at 3p14 that encompasses the FHIT and PTPRG genes, was also found in three samples of benign proliferative breast disease associated with familial BRC (18). Each of the deleted regions was considered as the possible site of one or several tumor suppressor genes.

Our previous work has shown that certain human 3p segments were regularly lost from chr3/A9 mouse fibrosarcoma MCHs in the course of tumor growth in SCID mice (19, 20). The same segments were frequently deleted in a variety of cancer-derived cell lines(20). We have proposed that the elimination of chromosome regions from monochromosomal hybrids during tumor growth can indicate the location of tumor antagonizing genes (19, 21). We have identified a CER at 3p22–3p21.3, between D3S1029 and D3S643/D3F15S2, that was lost from all of the 27 SCID tumors derived from 5 different MCHs (20). The region was gradually reduced to 1 Mb and designated CER1 (22, 23). No HDs were reported within CER1 in human tumors. A second eliminated region (ER2) was found at 3p21.1–3p14.2, between D3S1235and D3S1067. ER2 includes markers, D3S2, ALAS1,D3S1578, D3S1289 and D3S1076. It was lost in 21 tumors that grew from 4 MCHs, but not in 6 tumors derived from the del(3)(pter–p21.1) carrying MCH910.7 (Fig. 2). ER2 borders on the region of frequent HDs within 3p14.2 that includes the FHITgene (20; see 24 for review). The HDs found in two SCLC lines (1, 3) overlap with ER2.

In the present work, we examine the question of whether known chr3p genes that reside within HDs found in SCLC, NSCLC, BRC, CC, GC, and cervical cancer lines or within CER1 (Fig. 1; Table 1), change their expression upon SCID passage. We have chosen five genes within the HD regions, ITGA4L at 3p22–3p21.3, LUCA1 at 3p21.3, PTPRG at 3p14.2, FHIT at 3p14.2, and DUTT1 at 3p13–3p12. We also examined 5 genes within CER1 at 3p21.3, LIMD1, CCR1, CCR2, CCR3, and CCR5. Four chr3p genes outside these regions, VHL at 3p26–3p25, MLH1 at 3p23–3p22, TGM4 at 3p22–3p21.33, and UBE1L at 3p21.3–3p21.2, were chosen as controls (Fig. 1 and Table 1).

Human monochromosome/mouse MCHs were generated by microcell-mediated chromosome transfer as described previously (25). The A9 mouse fibrosarcoma cell line served as recipient. Four MCHs that carried chr3 were used for this study (Figs. 1 and 2). In the case of MCH910.61, MCH910.7 (19), and A9–3Neo, the chromosome donors were neomycin-resistant clones of normal human diploid fibroblast lines that were randomly tagged with the pSV2neo marker. The MCH line A9Hytk3 contained intact chr3 tagged with the bacterial hygromycin phosphotransferase (Hy) and herpes simplex virus thymidine kinase (tk) genes (26). The MCHs were maintained in growth medium containing geneticin (G418; Sigma, St. Louis, MO) at a final concentration of 400 μg/ml or hygromycin B(Calbiochem Corp., San Diego, CA) at a final concentration of 400 units/ml. Cells were injected s.c. into SCID mice(10⁵ cells/mouse). All of the tumors were explanted and cultured in vitro for 2–4 passages to obtain the necessary cell number for cytogenetic and molecular analyses.

Chromosome painting with chr3-specific probe was performed as described previously (19). FITC antidigoxigenin or rhodamine (Rh)antiavidin were used for immunochemical detection. A 4,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI)counterstaining was applied to visualize G- or R- bands. FISH and image analyses were performed using a fluorescent microscope (LEITZ-DMRB;Leica, Heidelberg, Germany) equipped with a Hamamatsu 4800 cooled CCD camera (Hamamatsu, Herrsching, Germany) and processing software Image-Pro Plus (Media Cybernetics) and Adobe Photoshop. A minimum of 20 metaphase cells was examined for each of the samples hybridized. More than 80% of the examined cells showed an identical pattern for each of the analyzed samples.

High-molecular-weight DNA was prepared by proteinase K digestion and phenol/chloroform extraction according to standard procedures(27). The PCR markers used for the genomic analysis are shown in Table 2. Genomic PCR was carried out using 200 ng of DNA as a template. Thirty cycles of 30 s at 94°C, 60 s at 50–55°C, and 2 min at 72°C, and a final extension of 5 min at 72°C followed initial denaturation at 95°C for 5 min. The PCR products were evaluated by electrophoresis through 2–3% agarose gels, stained with ethidium bromide, and photographed. Abnormal results were confirmed by duplex RT-PCR using two pairs of primers simultaneously.

RNA was extracted from the MCH and tumor cell lines using TRIzol (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s protocol. Random-primed, first-strand cDNA were synthesized from 6 μg of DNaseI-treated total RNA in a 50-μl volume using Superscript II(Life Technologies, Inc., Grand Island, NY) according to the instructions. Each cDNA synthesis reaction was paired with a control reaction without the addition of reverse transcriptase. The synthesized cDNA and the controls were aliquoted (5 μl/tube) and stored at−80°C.

Five μl of each synthesized cDNA and the controls (RNA) were subjected to PCR in a volume of 25 μl with the same PCR conditions and cycling as described for the genomic PCR. Primers used for RT-PCR analysis are shown in Table 1. Low-stringency PCR (at 45°C of annealing) was carried out to detect the full-length FHITtranscript and a truncated FHIT transcript. To exclude RNA degradation as an explanation for the absence of FHIT expression, duplex RT-PCR using two pairs of primers simultaneously, was performed.

Two new MCHs, A9–3Neo and A9Hytk3, and a subline of the previously tested MCH910.6, designated MCH910.61, carried cytogenetically normal chr3, derived from three different donors. A fourth previously studied line, MCH910.7 carried del(3)(pter–p21.1); (Fig. 2). The recipient A9 mouse fibrosarcoma line and all MCHs were tumorigenic in SCID mice, with take incidences over 80%. Thirteen SCID passaged tumors were collected, three from each MCH that carried a cytogenetically normal chr3, and four from MCH910.7.

The MCH lines and two derived SCID tumors from each were analyzed by FISH-P (Table 3). We have previously shown that the introduced chromosomes were maintained in the MCHs without significant change during at least 60 days of in vitro cultivation in the absence of G418 (19). We have also found that SCID tumors derived from MCH910.7 and MCH939.2 that carried del(3)(pter–p21.3) and del(3)(p21.3–p14), respectively,retained the introduced chromosome in its original form (Fig. 2). In contrast, MCHs that carried a normal chr3, had lost the entire introduced chromosome in 7 of 19 MCH906.8- and MCH910.6-derived tumors,or maintained parts of it with chimeric translocations in 12 of 19 MCH903.1- and MCH906.8-derived tumors (Fig. 2).

In the present study, we found that the MCH910.61 line carries a cytogenetically normal chr3 in 65% of the cells whereas 30% of the cells contain chimeric translocations. Derived tumors contained translocations in 42–45% of the cells and a cytogenetically intact chr3 in 2–6% of the cells (Table 3). The MCH A9–3Neo line carries a cytogenetically normal chr3 in 98% of cells, with no translocations. Chimeric translocations were not found in the derived tumors either. A cytogenetically intact chr3 was maintained in T3 and T1 tumors in 16 and 48% of the cells, respectively (Table 3). In contrast, the MCH A9Hytk3 line that carried a cytogenetically normal chr3 in 96% of the cells, has produced after SCID passage numerous chimeric translocations (found in T1 and T3 tumors in 24 and 12% of the cells,respectively). The cytogenetically intact chr3 was still present in 58% of the tumor T1 and 75% of the tumor T3 cells (Table 3). The del(3)(pter–p21.3) chromosome was present in 98% of the cells in the MCH910.7 in vitro line and was maintained in 35–37%of the cells in the derived SCID tumors.

Thirteen SCID tumors were analyzed by PCR, using 72 chr3p-specific and 11 3q-specific markers (Table 2). All of the tumors that were derived from MCH910.61, MCH A9–3Neo, and MCH A9Hytk3, were PCR-positive for all of the markers tested. They will be referred to as “PCR+”tumors. In the MCH910.61-derived tumors, the markers D3S1547,D3S1234, and D3S1300, located at 3p14.2, amplified a reduced amount of the product (data not shown). The FHIT-ex6 primers(from the exon 6 of FHIT) did not yield any PCR product in any of the MCH910.61-derived tumors (three were examined), in contrast to the normal amplification in all of the three MCH A9–3Neo-derived tumors, three MCH A9Hytk3 tumors, and four MCH910.7-derived tumors(Fig. 3,B). The MCH910.7 line that carried del(3)(pter–p21.3), and all derived tumors were negative for all of the PCR-markers from D3S3888 to the telomere. The duplex-PCR is exemplified in Fig. 3, B and C. Taken together with our previously published data, 24 of the 34 MCH-derived tumors were negative for at least one FHIT PCR marker. Ten of the 34 tumors retained all of the FHITmarkers tested (Fig. 2).

Fig. 1 summarizes the expression of the selected chr3p genes in the MCH lines and derived PCR+ tumors. Only one of the 14 genes studied, FHIT, showed a consistent down-regulation after SCID passage. The three MCH910.61-derived tumors have lost the genomic FHIT sequence (Fig. 3,B). No FHITtranscripts were found in any of the MCH A9–3Neo- and MCH A9Hytk3-derived tumors (Figs. 1 and 3,A). A truncated FHIT mRNA was found together with a reduced level of the full-length FHIT transcript in four tumors derived from MCH910.7 that carried del(3)(pter–p21.3). The truncated FHIT transcript was already present in the in vitro propagated MCH910.7 line (Figs. 1 and 3,E). In our previous experiments (19, 20) we found that the del(3)(pter–p21.3) in MCH910.7 and the del(3)(p21.3–p14) in MCH939.2 were retained in their original intact form after SCID passage. The FHIT transcript was not found in the MCH939.2 line in vitro (Fig. 3,A), in derived tumors, and in two MCH906.8-derived tumors that have maintained the FHIT region at 3p14.2 (Figs. 1 and 2). Small lesions (inversions, insertions, and deletions) within the gene might hamper FHIT expression, as reported for a variety of human tumors (see Ref. 24 for review). (The mRNA expression of other chr3p genes was not analyzed in these tumors, because of the lack of the material).

PTPRG was expressed in the MCH910.61 line but not in three derived tumors. Ten other tumors derived from the three other MCHs maintained their PTPRG expression (Fig. 1). VHL, MLH1, ITGA4L, UBE1L, LUCA1,and DUTT1 were expressed in the parental MCHs and in derived PCR+ tumors as well (Figs. 1 and 3,A). MCH910.61-derived tumors showed a significantly reduced mRNA expression of UBE1L and LUCA1, localized in the 3p21.3–p21.2 region (Fig. 1). The recently identified LIMD1 gene, located within CER1 (28), showed a reduced RNA expression in the MCH910.61-derived tumors and a total loss of expression in the MCH A9Hytk3-derived tumors. However, the LIMD1 transcript was present in the MCH A9–3Neo-derived tumors at the same level as in the parental MCH line (Fig. 1). TGM4, CCR1, CCR2, CCR3, and CCR5 mRNA was not detected in any of the MCHs or derived tumors by one-step RT-PCR(Figs. 1 and 3 D).

The FHIT gene, cloned in 1996, includes FRA3B, the most common fragile site at 3p14.2 and the hereditary renal cancer t(3;8) translocation breakpoint(12). The gene covers approximately 1 Mb and encodes a 1.1-kb transcript with 10 small exons. Exon 5 is the first protein-coding exon. It is flanked by FRA3B in intron 4 and intron 5. The M_r 16,800 Fhit protein hydrolyzes diadenosine triphosphate (ApppA) to ADP and AMP in vitro. Mutation of a central histidine abolishes hydrolytic activity (see Ref. 29 for review). Using RT-PCR and cDNA sequence analysis, Ohta et al. (12) have detected aberrant FHIT transcripts in 13 of 27 uncultured esophagus, stomach, and colon tumors. Normal-sized transcripts were also observed in 8 of the 13 tumors with aberrant transcripts. Aberrant FHIT transcripts have also been found in 30% of breast carcinomas (30), 80% of primary SCLCs, 40% of NSCLCs(31), and 55% of squamous cell carcinomas of the head and neck (32). Six of 7 cervical carcinoma lines showed no or reduced FHIT expression (33). Aberrant FHIT RNA expression correlated with the lack of the detectable Fhit protein in 10 cancer-derived cell lines of the kidney,colon, stomach, cervix, head and neck, and nasopharynx(34). Expression of exogenous Fhit protein in four FHIT-negative cancer cell lines that originated from different tumors, was found to abrogate their tumorigenicity in nude mice (35).

In the present study, we have tested human chr3/A9 mouse fibrosarcoma MCH-derived SCID tumors that have maintained the entire chr3(derivatives of MCH910.61, MCH A9–3Neo, and MCH A9Hytk3) or the del(3)(pter–p21.3) (derivatives of MCH910.7), for the expression of human chr3p-genes. All of the five MCH lines that contained an intact chr3p, expressed FHIT in vitro. In all of the analyzed tumors, FHIT was functionally impaired. Nine PCR+tumors derived from three MCHs (three from each), MCH910.61, MCH A9–3Neo, and MCH A9Hytk3, failed to express FHIT. Three tumors generated from MCH910.61 revealed a deletion including exon 6 of FHIT. Four tumors derived from the MCH910.7 line expressed a truncated mRNA, in parallel with the normal-sized transcript, detected at a reduced level. Five of the 14 examined chr3p-genes, TGM4, CCR1, CCR2, CCR3, and CCR5, were not expressed in the MCH lines in vitro. Eight genes, VHL, MLH1, ITGA4L, LIMD1, UBE1L, LUCA1, PTPRG, and DUTT1, were expressed in the MCHs in vitro and in all derived tumors. The down-regulation of FHIT expression after SCID mouse passage contrasts, moreover, with the maintained expression of eight other genes, localized in or near regions that are targets of tumor-associated HDs.

The validity of the A9-based MCH elimination system for the functional detection of tumor suppressor genes is also supported by the finding of Li et al. at our Center (Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden; Ref.36) showing that the human RB gene, transfected into A9 cells, is either eliminated or down-regulated, after SCID mouse passage. The elimination test provides a possibility to detect malignancy suppressors in frequently deleted, but otherwise unknown,areas of the genome. Meanwhile, FHIT qualifies for the role of a malignancy suppressor, as also indicated by the suppression study of Siprashvili et al. (35).

We thank Dr. J. Carl Barrett (National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA) and Dr. J. D. Hunt (Louisiana State University Medical Center, New Orleans, LA) for supplying us with the cell line A9–3Neo; Drs. Robert F. Newbold and Andrew P. Cuthbert (Brunel University, Uxbridge, United Kingdom) for supplying us with the cell line A9Hytk3; and Dr. Eric G. Stanbridge (University of California, Irvine, CA) for the MCHs MCH903.1, MCH906.8, MCH910.6, MCH939.2, and MCH910.7. We thank Lena Norenius, Agneta Sandlund, May-Lis Solberg, and Margareta Hagelin for technical assistance.