Affiliations of authors: R. Wu, D. C. Connolly, K. R. Cho, Departments of Pathology and Internal Medicine, The University of Michigan Medical School, Ann Arbor; R. L. Dunn, Biostatistics Core, University of Michigan Comprehensive Cancer Center, Ann Arbor.
Correspondence to: Kathleen R. Cho, M.D., Department of Pathology, The University of Michigan Medical School, 4301 MSRBIII, 1150 W. Medical Center Dr., Ann Arbor, MI 48109 (e-mail: kathcho{at} umich.edu).
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ABSTRACT |
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INTRODUCTION |
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In 1996, a candidate tumor suppressor gene named FHIT (fragile histidine triad) was identified at chromosome 3p14.2 (9). FHIT is a large gene, with at least 10 exons spanning approximately 1 megabase of genomic DNA. The coding region, beginning in exon 5 and ending in exon 9, encodes a protein with a predicted molecular mass of approximately 16.8 kd. Fhit protein has been shown to function as a dinucleoside 5',5'''-P1,P3-triphosphate (Ap3A) hydrolase, forming adenosine diphosphate and adenosine monophosphate from the Ap3A substrate (10). Several published reviews (11) have summarized observations supporting FHIT's candidacy as a tumor suppressor gene targeted by 3p14 allelic losses in cervical and several other types of human cancers. For example, the t(3;8) (p14.2;q24) translocation in a kindred with familial renal cell carcinoma occurs within the FHIT locus, and homozygous deletions within the FHIT gene, some of which include coding-region exons, have been identified in several primary tumors and cell lines. In addition, aberrant FHIT transcripts have been observed in many of the different tumor types that show frequent deletions of the 3p14.2 region. More recent studies (15) suggest a role for Fhit in the regulation of apoptosis and the cell cycle.
While these findings support FHIT's candidacy as a tumor suppressor gene, other findings question this view. The FHIT locus spans a large common fragile site/region, FRA3B, and it has been suggested that structural alterations within FHIT simply reflect genetic instability of FRA3B. Moreover, aberrant FHIT transcripts of the type seen in cancers have been observed in non-neoplastic tissues, point mutations within the FHIT coding region are extremely rare, and some homozygous deletions affect only FHIT introns and leave the exons intact. Functional studies assessing Fhit's ability to suppress the tumorigenic properties of cancer cells have also yielded conflicting results. Two groups (16,17) have reported that overexpression of Fhit suppresses the tumorigenicity of selected Fhit-negative lung carcinoma cell lines. Tumor suppression by Fhit has also been observed in gastric and renal carcinoma-derived cell lines (17). In contrast, Otterson et al. (18) reported that Fhit protein expression failed to suppress tumorigenicity of HeLa cervical carcinoma cells. This group of authors suggested that the contradictory results raise the possibility that FHIT exerts its tumor suppressive activity in a highly cell lineage-specific manner.
We have previously characterized FHIT expression in six cervical cancer cell lines, in 35 primary cervical carcinomas, and in various normal tissues, including normal cervix (19), and found aberrant FHIT transcripts and markedly reduced or absent Fhit protein expression in about 70% of primary cervical carcinomas. Others (20-23) have reported aberrant FHIT transcripts and/or loss of Fhit protein expression in 43%-61% of invasive cervical cancers and a third of high-grade cervical intraepithelial lesions but only rarely in low-grade cervical intraepithelial lesions (22,23). Integration of HPV into the FRA3B/FHIT locus has also been observed in a primary cervical carcinoma (24). Collectively, the findings suggest that FHIT gene alterations may play an important role in cervical tumorigenesis. In this study, we sought to further define the role of FHIT in cervical cancer by restoring Fhit expression in selected cervical and other cancer cell lines.
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MATERIALS AND METHODS |
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Vector construction. Full-length, wild-type FHIT complementary DNA (cDNA) was inserted into the EcoRI site of the pPGS-CMV-CITE- Neo(+) plasmid (provided by G. Nabel, University of Michigan, Ann Arbor), which contains the retroviral long terminal repeat and packaging signal for efficient expression. Chimeric transcripts encoding the gene of interest fused to the neomycin resistance gene are generated from this vector. An internal ribosomal entry site allows translation of both proteins from the chimeric transcript. The FHIT cDNA was also subcloned into the EcoRI site of pcDNA3.1/zeo+ vector (Invitrogen Corp., Omar, UT) to generate pcDNA3.1/zeo+/FHIT, in which FHIT expression was under the transcriptional control of a strong constitutive cytomegalovirus (CMV) promoter. The full-length FHIT inserts of both plasmids were verified by sequence analysis.
Retrovirus infection. To generate recombinant retroviruses, we transfected amphotropic Phoenix cells with the retroviral expression plasmids by use of the FuGene6TM nonliposomal transfection reagent (Boehringer Mannheim Biochemicals, Inc., Indianapolis, IN), as described by the manufacturer. Briefly, 2.5 x 106 Phoenix cells were seeded in 60-mm dishes 12 hours before transfection. Cells were then transfected with 6 µg of pPGS-CMV-CITE- Neo(+)/FHIT or pPGS-CMV-CITE-Neo(+) and 15 µg of FuGene6. Forty-eight hours after transfection, culture supernatants containing the retroviruses were added to the C-33A, C-4II, H460, and AGS cells seeded at a density of 2 x 106/25-cm2 flask the preceding day. Cells were initially selected in 1 mg/mL G418 (Geneticin; Life Technologies, Inc.) starting 48 hours after infection. G418 concentration was reduced to 0.7 mg/mL and then to 0.25 mg/mL 5 and 10 days after retroviral infection, respectively. Polyclonal populations of stably transduced cells were evaluated for Fhit protein expression approximately 2 weeks (two passages) following transduction and injected into nude mice three passages after transduction.
Stable transfection. Transfections were performed by use of Lipofectace transfection reagent (Life Technologies, Inc.) with 2 µg of pcDNA3.1/zeo+/FHIT or pcDNA3.1/zeo+. Plasmids were added to 25-cm2 flasks containing C-33A cells seeded at a density of 3 x 105 cells the preceding day. Clones were selected in the presence of 250 µg/mL zeocin (Invitrogen Corp.). Clonal sublines of C-33A transfectants were obtained by limiting dilution. Individual clones were initially evaluated for Fhit protein expression 3 months following transfection (passage 4) and again at passage 7. Cells were injected into nude mice at passage 7.
Western blot analysis. Lysates from cultured cells were prepared in lysis buffer composed of 5 mL 1% sodium dodecyl sulfate (SDS)/10 mM Tris (pH 7.4) and one tablet CompleteTM protease inhibitor cocktail (Boehringer Mannheim Biochemicals). Xenograft tissues were homogenized in lysis buffer, and the supernatants were prepared by centrifugation at 13 000g for 30 minutes at 4 °C. Protein concentrations were measured by the bicinchoninic acid protein standard method (Pierce Chemical Co., Rockford, IL). Equivalent amounts (30-100 µg) of protein from each sample were loaded on 12% resolving polyacrylamide gels and then transferred to Immobilon-P membrane (Millipore Corp., Marlborough, MA). Fhit protein was detected with an anti-gst-Fhit rabbit polyclonal antibody (provided by K. Huebner) at a dilution of 1 : 2500 (27). Detection of antigen-antibody complexes was carried out with an enhanced chemiluminescence detection kit (Amersham Life Science Inc., Arlington Heights, IL) per the manufacturer's protocol.
Southern blot analysis. To determine the independence of C-33A clonal sublines transfected with pcDNA3.1/zeo+/FHIT or pcDNA3.1/zeo+, Southern blot analyses were performed. Genomic DNA was isolated from cultured cells by use of SDS/proteinase K digestion followed by phenol/chloroform extraction. Ten micrograms of each DNA sample was digested with 100 U of EcoRI at 37 °C overnight. The digested DNA samples were separated by agarose gel electrophoresis, then transferred to Zeta probe membrane (Bio-Rad Laboratories, Inc., Hercules, CA). Hybridization to a 32P-labeled pcDNA3.1/FHIT probe was carried out in Rapid Hyb Solution (Amersham Life Science Inc.), as recommended by the manufacturer. The membranes were exposed to XAR5 autoradiography film (Kodak, Rochester, NY) for varying lengths of time at -80 °C. Several clones revealing unique band patterns were selected for further analysis.
Colony formation in soft agar. Dishes (35 mm) were precoated with 1 mL of 0.6% agar/1x complete medium (medium with FBS). Suspensions of 104 cells in 0.3% agar/1x complete medium were spread over precoated wells and fed twice per week by adding small amounts of growth medium. Each experiment was performed in duplicate. Cultures were incubated for 4 weeks, then fixed and stained with 1.5% glutaraldehyde/0.06% methylene blue (vol/wt) solution. The total number of colonies in each dish greater than 100 µm in diameter was counted with the aid of a microscope eyepiece containing a grid.
Tumorigenicity in nude mice. Polyclonal populations of C-33A, C-4II, and H460 cells transduced by use of recombinant retroviruses with and without the FHIT cDNA insert and several independent clones of C-33A cells stably transfected with pcDNA 3.1/zeo+/FHIT or pcDNA 3.1/zeo+ vector alone were evaluated for tumorigenicity in nude mice. Six-week-old female athymic nude (nu/nu) mice (Charles River Laboratories, Wilmington, MA) were given subcutaneous injections in the left and right flanks of 5 x 106 cells in 0.2-mL Hanks' balanced salt solution without phenol red (Life Technologies, Inc.). Cells transfected/transduced with each construct were injected into five mice, which were ear-tagged and followed individually throughout the study. Mice were examined two to three times per week for tumor formation at the sites of injection. In situ tumor measurements were taken at least once per week starting on day 7. Experiments were terminated 30 days (C-33A and C4II) or 2 weeks (H460) after injection. H460 xenografts were harvested earlier, since previous experiments with the parental cells showed that growth for longer periods of time resulted in significant tumor necrosis. Animals were killed with CO2, then examined for tumor growth at the injection sites and for tumor dissemination. Tumors were weighed and measured with linear calipers, then divided into several portions for further analyses. Representative samples of each tumor were fixed in 10% buffered formalin, routinely processed, and embedded in paraffin. Tissue sections (5 µm) from representative xenografts were cut and mounted on superfrost Plus slides (Fisher, Itasca, IL) and stained with hematoxylin-eosin for histopathologic evaluation or used for immunohistochemistry. Representative portions of xenograft tissues were also snap-frozen in liquid nitrogen and stored at -80 °C until utilized as a source of protein for western blot analysis. All animal studies were conducted in accordance with a study protocol approved by the University of Michigan's Committee on Use and Care of Animals.
Immunofluorescent staining of Fhit protein. Approximately 105 cultured cells were seeded on 3-aminopropyltriethoxysilane-treated slides (Digene Diagnostics, Inc., Beltsville, MD), grown for 2 days, fixed in 4% paraformaldehyde, then permeabilized in 1% goat serum/0.5% Triton X-100/phosphate-buffered saline (PBS). After washing with PBS, the slides were blocked with 20% goat serum for 30 minutes at room temperature. Samples were incubated with the rabbit polyclonal anti-Fhit antibody at 1 : 200 dilution for 1 hour at room temperature. Slides were washed three times in PBS, then incubated with Texas Red-conjugated goat anti-rabbit immunoglobulin G (Jackson ImmunoResearch Lab Inc., West Grove, PA) at a dilution of 1 : 150 for 30 minutes at room temperature. The stained cells were visualized under a fluorescence microscope (Olympus, Melville, NY).
Immunohistochemical staining of Fhit protein. Tissue sections (5 µm) were dewaxed in xylene, then rehydrated through graded alcohols. Antigen retrieval was performed by boiling slides in citrate buffer (pH 6.0; Biogenex, San Ramon, CA) in a microwave oven for 10 minutes. Endogenous peroxidase activity was blocked by incubation with 6% hydrogen peroxide in methanol. Slides were then incubated with primary antibody (polyclonal rabbit gst-Fhit antiserum) at a dilution of 1 : 4000 overnight at 4 °C. After washing in PBS, slides were incubated with a biotinylated goat anti-rabbit secondary antibody for 30 minutes at room temperature. Antigen-antibody complexes were detected with the avidin-biotin-peroxidase method by use of diaminobenzidine as a chromogenic substrate (Vectastain ABC-Immunostaining Kit; Vector Laboratories, Inc., Burlingame, CA), as recommended by the manufacturer. Tissue sections were lightly counterstained with hematoxylin, then evaluated by light microscopy. Xenograft tissues derived from C-33A parental cells were used as negative controls in each staining assay. We have demonstrated previously specificity of the polyclonal anti-Fhit antibody in immunohistochemical applications (19).
Statistical methods. For the experiments involving colony formation in soft agar, the number of colonies in each dish more than 100 µm were counted. Separate counts were recorded for colonies 100-200 µm, 201-300 µm, and greater than 300 µm. The remainder of the initial group of 104 cells were placed into the less than 100-µm category. Each experiment was performed in duplicate, and the data from both experiments were combined for the analysis. Two separate analyses were performed on these data. First, a Mantel-Haenszel chi-squared test was used to test for differences in the linear relationship with the size of the colony between the FHIT group and the control group. Second, a general chi-squared test was used to test for differences in the proportion of colonies that were at least 100 µm between the FHIT and control groups. Each analysis was performed separately by cell line. For the experiments in which tumorigenicity in nude mice was studied, transfected cells were injected into the left and right flanks of five mice per cell line. The experiments were terminated at the specified time points, and the tumors were excised. Mixed models were used to test for differences in the tumor volume and weight between the FHIT and the control groups. This analysis adequately adjusted for the association inherent in the data because of multiple tumors being measured from the same mouse. Separate models were created for each cell line. For the C33A cells, a separate analysis was also performed that included the data from the clonal sublines. In the analysis, clones were treated identically to the polyclonal cell populations.
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RESULTS |
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We evaluated several cervical carcinoma cell lines by immunoblot
analysis and confirmed the absence of endogenous Fhit protein
expression in two cell lines, C-33A and C-4II, previously shown to lack
full-length FHIT transcripts (19). C-33A has a homozygous
deletion within FHIT, including exon 5 (4), and C4I, a cell
line derived from the same cancer specimen as C-4II, has also been
reported to have a homozygous deletion within the FHIT locus
(28). As expected, Fhit protein was not detected by western
blot analysis in either C-33A or C-4II cervical carcinoma cells (Fig.
1, A). Fhit protein of the expected size was
detected in HeLa, CaSki, SiHa, and ME180 cervical cancer cells, as well
as in primary foreskin keratinocytes (Fig. 1,
A). Our detection of
full-length FHIT transcripts and Fhit protein in HeLa contrasts with
other studies in which Fhit protein was reported to be undetectable
(18). The different observations of Fhit expression in HeLa
likely reflect the genetic variability of HeLa sublines that have been
used by different laboratories (20,27). C-33A and C-4II cell
lines are tumorigenic in nude mice, forming poorly differentiated
squamous carcinomas on subcutaneous injection into the flank. H460
cells, derived from a large-cell carcinoma of the lung, also form
poorly differentiated tumors in nude mice. Previous studies
(17,29) have shown that H460 cells harbor homozygous deletion
of FHIT (intron 5) and lack expression of endogenous Fhit protein.
Western blot analysis confirmed lack of Fhit protein expression in H460
cells (Fig. 1,
B) and AGS cells (data not shown). In our studies, the
injection of up to 1 x 107 AGS gastric carcinoma cells
failed to generate tumors in nude mice during a 9-week surveillance
period. AGS cells were, therefore, excluded from further studies of
Fhit's ability to suppress their tumorigenic growth in nude mice but
included in our analysis of Fhit's effects on anchorage-independent growth.
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Recombinant retroviruses carrying the cloned FHIT cDNA as well as a
G418 selectable marker were used to transduce C-33A, C-4II, and H460
cells. Fhit protein of the expected size was easily detected in
polyclonal populations of cells infected with retrovirus carrying FHIT
and in the derivative tumor xenografts (Fig. 1, B, lanes 1-6) but not
in cells infected with a retrovirus lacking the FHIT cDNA insert (Fig.
1,
B, lanes 7-9). Expression of Fhit protein was also verified in six
of 14 zeocin-resistant clones of C-33A cells stably transfected with
the pcDNA3.1/zeo+/FHIT expression plasmidthree representative clones
[C-33A-F(CL1, CL2, and CL3)] shown in Fig. 1,
C, lanes 4-6.
Four of
the six pcDNA clones, including the three shown in Fig. 1,
C, were
shown to be independent by Southern blot analysis (data not shown).
Fhit protein expression was not detected in any of 12 C-33A clones
transfected with the pcDNA3.1/zeo+ vector alonerepresentative clone
[C-33A-V(CL-1)] shown in Fig. 1,
C, lane 3. Expression of
Fhit
protein in C-33A, C-4II, and H460 cells transduced by recombinant
retroviruses was also confirmed by immunofluorescence staining prior to
further analysis (Fig. 2, A-C)
. Some heterogeneity
of Fhit expression was noted, with 30%-40% of stably transduced
cells expressing very high protein levels and the remainder expressing
lower, but detectable, amounts of Fhit protein. Fhit expression was not
detected by immunofluorescence staining in control (empty vector)
C-33A, C-4II, or H460 cells (data not shown). No obvious changes in
cellular morphology or growth kinetics were noted in cells expressing
Fhit protein.
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The parental cell lines were subcutaneously injected into nude mice
to confirm their tumorigenic growth properties. Injection of 5 x
106 cells from the C-33A and C-4II cervical cancer lines and
the H460 lung cancer line resulted in formation of palpable tumors
after 2 weeks. Stably selected polyclonal populations of each cell line
infected with either recombinant FHIT or control retroviruses were
injected into groups of five mice, so that each population of cells was
evaluated at 10 injection sites. A total of six independent, stably
transfected C-33A clones [three transfected with pcDNA 3.1/zeo+/FHIT
(C-33A F1-3) and three transfected with vector alone (C-33A V1-3)]
were also evaluated. Expression of Fhit failed to suppress tumor
formation within the surveillance period in any of the polyclonal or
clonal cell lines tested. The net weights and volumes of tumor tissues
obtained immediately after excision from euthanized mice are shown in
Fig. 3, A-D. There was no statistically significant
difference in the weight or volume of tumors formed by polyclonal
populations of retrovirally transduced C-33A, C-4II, or H460 cells
expressing Fhit versus control (P>.05; mixed models; Fig.
3,
A and B). Similarly, there was no statistically significant
difference in the weight or volume of tumors formed by clonal
populations of C33A cells transfected with the pcDNA3.1/zeo+/FHIT
plasmid versus vector control (P>.05; mixed models; Fig. 3,
C and D). The tumors formed by parental and vector-only transduced
C-4II cells were rather small, with in vivo regression
observed starting 2 weeks after injection. Tumor formation by C-4II
cells was documented by microscopic examination of xenograft tissues,
and no differences were noted between Fhit-positive and the
Fhit-negative cells. Tumor xenografts formed by Fhit-expressing C-33A,
C-4II, and H460 cells were shown to retain strong Fhit protein
expression by both immunoblot and immunohistochemical analyses. Fig. 1,
B (lanes 2, 4, and 6), shows retention of Fhit expression in xenografts
derived from polyclonal populations of cells transduced by recombinant
retroviruses. Fig. 1,
C (lanes 7 and 8), shows retention of Fhit
expression in xenografts derived from clonal populations of C-33A cells
stably transfected with the pcDNA3.1/zeo+/FHIT plasmid.
Immunohistochemical detection of Fhit protein expression in
representative tumor xenografts is shown in Fig. 2,
D-L. Panels D and
E show Fhit expression in a xenograft derived from a clonal population
of C-33A cells (C-33A-F1) stably transfected with pcDNA3.1/zeo+/FHIT.
Panels F and G show Fhit expression in a xenograft derived from
polyclonal C-33A cells transduced with the
pPGS-CMV- CITE-Neo(+)/FHIT recombinant retrovirus. Panels H
and I show Fhit expression in retrovirally transduced C-4II and H460
cells, respectively. Roughly comparable heterogeneity of Fhit protein
expression was noted in preinjection cells and xenografts, with the
xenografts retaining a substantial number of cells strongly expressing
Fhit. For comparison purposes, the absence of Fhit expression in
representative xenografts formed by polyclonal populations of C-33A,
C-4II, and H460 cells retrovirally transduced with vector alone is
shown in panels J-L.
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As shown in Table 1, cells with and without
Fhit expression were examined for alterations in anchorage-independent
growth properties. C-4II and AGS cells showed no difference in the
number or size of colonies formed by cells expressing Fhit versus
vector control (P>.05). However, C-33A and H460 cells
expressing Fhit formed more and larger colonies than cells transduced
with vector alone (all P = .001). This result suggests that,
under certain conditions, Fhit expression may actually increase the
anchorage-independent growth properties of selected cancer cells. A
similar observation was made by Siprashvili et al. (17), who
noted that AGS and RC48 cells formed larger colonies compared with
control cells.
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DISCUSSION |
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Studies of Fhit's ability to suppress tumorigenicity of cancer cells continue to yield
conflicting
results. Collectively, we and Otterson et al. have demonstrated Fhit's inability to suppress
tumorigenicity of three different cervical carcinoma cell lines (HeLa, C-33A, and C4II). If, as
Otterson
et al. have suggested, Fhit exerts its tumor-suppressive effect in a cell lineage-specific manner (18), Fhit may well suppress tumorigenicity of cell lines derived from lung,
kidney, or digestive tract cancers but not those derived from cervical cancers. However, this
explanation cannot account for the different results obtained by three independent groups studying
H460 lung carcinoma cells. Genetic heterogeneity of H460 sublines utilized in different
laboratories may
be, in part, responsible for the different outcomes because genetic variation at the FHIT locus has
been
described in other cancer cell sublines such as HeLa (27,30). The
contradictory results may also reflect differences in the level and degree of heterogeneity of
exogenous
Fhit expression obtained in recipient cells (such as H460) by use of various gene delivery systems.
In
our hands, heterogeneous Fhit expression was observed when either retroviral or plasmid vectors
were
employed to express exogenous Fhit protein, even when clonal sublines of cells were isolated.
Other
investigators may have achieved more uniformly elevated levels of Fhit expression in the cells that
they
tested. However, if heterogeneity of Fhit expression is solely responsible for the different
outcomes, one
might expect a delay of tumor growth of Fhit-positive versus Fhit-negative control cells in
addition to
more uniform selection against Fhit-expressing cells in the xenografts. We made neither of these
observations. Direct comparison of Fhit expression and function in cells generated by the different
laboratories and with different constructs may well shed additional light on these problematic
results.
Notably, we found comparable levels of Fhit expression in clonal preinjection cells and their
derivative
xenografts, regardless of whether the cells initially expressed high [e.g.,
C-33A-F(CL2)]
versus low [C-33A-F(CL3)] levels of Fhit protein (Fig. 1, C).
Although our study fails to provide additional functional evidence supporting FHIT as a tumor suppressor gene, we wish to emphasize that our analysis by no means excludes FHIT as a suppressor gene candidate. In light of the presence of frequent 3p14 deletions, FHIT somatic mutations, and alterations of FHIT expression in cervical cancer precursor lesions, it is possible that FHIT inactivation may be critical early in cervical cancer development or progression. However, restoration of Fhit expression in cervical cancer cells that have accumulated numerous other genetic alterations may not be sufficient for demonstrably affecting their growth or tumorigenic properties. Clearly, additional studies will be required to fully understand the role of FHIT in human cancer.
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NOTES |
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We thank Eric R. Fearon for his helpful discussions and review of this manuscript.
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Manuscript received June 10, 1999; revised November 18, 1999; accepted December 6, 1999.
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