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Restored Expression of Fragile Histidine Triad Protein and Tumorigenicity of Cervical Carcinoma Cells

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Abstract

Background: Allelic losses in the short arm of chromosome 3 are common in cervical carcinomas. The fragile histidine triad (FHIT) gene at chromosome region 3p14.2 is a candidate tumor suppressor gene that may play a role in cervical tumorigenesis. We and others have identified aberrant FHIT transcripts and frequent loss of Fhit protein expression in primary cervical cancers and high-grade noninvasive lesions but not in normal cervical tissues. The altered expression of FHIT may be due to somatic mutations or integration of human papillomavirus DNA at the FHIT locus. The purpose of this study was to determine whether ectopic expression of Fhit can suppress the tumorigenic properties of cervical cancer cells. Methods: We employed infection with recombinant retroviruses as well as transfection of plasmid DNA to restore Fhit protein expression in cervical cancer cell lines lacking full-length FHIT transcripts and endogenous Fhit protein. The effects of Fhit expression on tumor cell morphology, anchorageindependent growth, and tumorigenicity in nude mice were examined. Results: Stable overexpression of Fhit had no discernible effect on the tumorigenic properties of two cervical carcinoma cell lines or on a lung carcinoma cell line previously reported by others to be suppressed for tumorigenicity by Fhit. Conclusions: Restoration of Fhit expression does not suppress anchorageindependent growth or tumorigenicity of cervical carcinoma cell lines. However, it remains possible that FHIT inactivation may be important early in cervical tumor progression or that FHIT may suppress tumorigenesis in ways distinct from those measured by the assays employed in this study. [J Natl Cancer Inst 2000;92:338‐44]
Restored Expression of
Fragile Histidine Triad
Protein and Tumorigenicity
of Cervical Carcinoma Cells
Rong Wu, Denise C. Connolly,
Rodney L. Dunn, Kathleen R. Cho
Background: Allelic losses in the short
arm of chromosome 3 are common in
cervical carcinomas. The fragile histi-
dine triad (FHIT) gene at chromosome
region 3p14.2 is a candidate tumor sup-
pressor gene that may play a role in
cervical tumorigenesis. We and others
have identified aberrant FHIT tran-
scripts and frequent loss of Fhit protein
expression in primary cervical cancers
and high-grade noninvasive lesions but
not in normal cervical tissues. The al-
tered expression of FHIT may be due to
somatic mutations or integration of hu-
man papillomavirus DNA at the FHIT
locus. The purpose of this study was to
determine whether ectopic expression
of Fhit can suppress the tumorigenic
properties of cervical cancer cells.
Methods: We employed infection with
recombinant retroviruses as well as
transfection of plasmid DNA to restore
Fhit protein expression in cervical can-
cer cell lines lacking full-length FHIT
transcripts and endogenous Fhit pro-
tein. The effects of Fhit expression on
tumor cell morphology, anchorage-
independent growth, and tumorigenic-
ity in nude mice were examined. Re-
sults: Stable overexpression of Fhit had
no discernible effect on the tumorigenic
properties of two cervical carcinoma
cell lines or on a lung carcinoma cell
line previously reported by others to be
suppressed for tumorigenicity by Fhit.
Conclusions: Restoration of Fhit ex-
pression does not suppress anchorage-
independent growth or tumorigenicity
of cervical carcinoma cell lines. How-
ever, it remains possible that FHIT in-
activation may be important early in
cervical tumor progression or that
FHIT may suppress tumorigenesis in
ways distinct from those measured by
the assays employed in this study. [J
Natl Cancer Inst 2000;92:338–44]
Cervical carcinoma remains a major
source of cancer-related morbidity and
death for women in the United States and
throughout the world. Infection with cer-
tain (“high-risk”) types of human papillo-
maviruses (HPVs) is strongly associated
with cervical cancer, probably due, in
large part, to inactivation of the cellular
tumor suppressor protein p53 and retino-
blastoma protein (pRB) through interac-
tions with the HPV E6 and E7 proteins,
respectively (1). Recent studies suggest
that, in addition to HPV infection, so-
matic mutations in oncogenes and tumor
suppressor genes are likely to be critical
for cervical cancer development and pro-
gression. Localization of tumor suppres-
sor genes inactivated during cervical tu-
morigenesis may be aided by the
identification of frequent allelic losses of
specific chromosomal regions in primary
tumors and associated precursor (intraep-
ithelial) lesions. Several groups of inves-
tigators (2–8) have demonstrated frequent
losses of heterozygosity on the short arm
of chromosome 3 in cervical cancers and/
or in preinvasive lesions. At least one
commonly deleted region has been
mapped to 3p14 (2–6).
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 ex-
ons spanning approximately 1 megabase
of genomic DNA. The coding region, be-
ginning in exon 5 and ending in exon 9,
encodes a protein with a predicted mo-
lecular mass of approximately 16.8 kd.
Fhit protein has been shown to function as
a dinucleoside 5,5-P
1
,P
3
-triphosphate
(Ap
3
A) hydrolase, forming adenosine di-
phosphate and adenosine monophosphate
from the Ap
3
A substrate (10). Several
published reviews (11–14) have summa-
rized observations supporting FHIT’s
candidacy as a tumor suppressor gene tar-
geted by 3p14 allelic losses in cervical
and several other types of human cancers.
For example, the t(3;8) (p14.2;q24) trans-
location in a kindred with familial renal
cell carcinoma occurs within the FHIT lo-
cus, and homozygous deletions within the
FHIT gene, some of which include cod-
ing-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
(15,16) suggest a role for Fhit in the regu-
lation 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 sug-
gested that structural alterations within
FHIT simply reflect genetic instability of
FRA3B. Moreover, aberrant FHIT tran-
scripts 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 ho-
mozygous deletions affect only FHIT in-
trons and leave the exons intact. Func-
tional 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 suppres-
sion 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 ex-
erts 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 carcino-
mas, and in various normal tissues, in-
cluding normal cervix (19), and found ab-
errant FHIT transcripts and markedly
reduced or absent Fhit protein expression
in about 70% of primary cervical carcino-
mas. Others (20–23) have reported aber-
rant FHIT transcripts and/or loss of Fhit
protein expression in 43%–61% of inva-
sive cervical cancers and a third of high-
grade cervical intraepithelial lesions but
only rarely in low-grade cervical intraep-
ithelial lesions (22,23). Integration of
HPV into the FRA3B/FHIT locus has also
been observed in a primary cervical car-
cinoma (24). Collectively, the findings
suggest that FHIT gene alterations may
play an important role in cervical tumori-
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., De-
partment of Pathology, The University of Michigan
Medical School, 4301 MSRBIII, 1150 W. Medical
Center Dr., Ann Arbor, MI 48109 (e-mail: kathcho@
umich.edu).
See “Notes” following “References.”
© Oxford University Press
338 REPORTS Journal of the National Cancer Institute, Vol. 92, No. 4, February 16, 2000
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genesis. 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.
MATERIALS AND METHODS
Cell lines. All human carcinoma cell lines were
obtained from the American Type Culture Collec-
tion (Manassas, VA). The cervical carcinoma-
derived cell lines HeLa, SiHa, C-33A, and C-4II
were cultured in Dulbecco’s modified Eagle me-
dium (DMEM)/10% (vol/vol) fetal bovine serum
(FBS). ME-180 cervical carcinoma cells were cul-
tured in McCoy’s 5A medium containing 10% FBS.
CaSki cervical carcinoma and H460 lung carcinoma
cells were cultured in RPMI-1640 medium/10%
FBS, and AGS gastric carcinoma cells were main-
tained in Ham’s F12 nutrient medium/10% FBS.
Media and FBS were obtained from Life Technolo-
gies, Inc. (GIBCO BRL), Gaithersburg, MD. Human
primary foreskin keratinocytes were cultured from
fresh neonatal foreskins and maintained in keratino-
cyte growth medium (Clonetics, Walkersville, MD),
as previously described (25). Amphotropic Phoenix
cells, which are highly transfectable 293T-derived
human embryonic kidney cells stably expressing the
MoLV retroviral gag, pol, and env genes, were used
to package and amplify recombinant retroviruses
[(26) and http://www.stanford.edu/group/nolan/NL-
phoenix.html]. These cells were maintained in
DMEM/10% FBS.
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 retro-
viral 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 con-
trol of a strong constitutive cytomegalovirus (CMV)
promoter. The full-length FHIT inserts of both plas-
mids 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 FuGene6™ nonliposomal transfection reagent
(Boehringer Mannheim Biochemicals, Inc., India-
napolis, IN), as described by the manufacturer.
Briefly, 2.5 × 10
6
Phoenix cells were seeded in 60-
mm dishes 12 hours before transfection. Cells were
then transfected with 6 g of pPGS−CMV−CITE−
Neo(+)/FHIT or pPGS−CMV−CITE−Neo(+) and 15
g 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 of2×10
6
/25-cm
2
flask the pre-
ceding 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. Poly-
clonal 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 trans-
duction.
Stable transfection. Transfections were per-
formed by use of Lipofectace transfection reagent
(Life Technologies, Inc.) with 2 g of pcDNA3.1/
zeo+/FHIT or pcDNA3.1/zeo+. Plasmids were
added to 25-cm
2
flasks containing C-33A cells
seeded at a density of 3 × 10
5
cells the preceding
day. Clones were selected in the presence of 250
g/mL zeocin (Invitrogen Corp.). Clonal sublines of
C-33A transfectants were obtained by limiting dilu-
tion. Individual clones were initially evaluated for
Fhit protein expression 3 months following transfec-
tion (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 mMTris
(pH 7.4) and one tablet Complete™ protease inhibi-
tor cocktail (Boehringer Mannheim Biochemicals).
Xenograft tissues were homogenized in lysis buffer,
and the supernatants were prepared by centrifuga-
tion at 13 000gfor 30 minutes at 4 °C. Protein con-
centrations were measured by the bicinchoninic acid
protein standard method (Pierce Chemical Co.,
Rockford, IL). Equivalent amounts (30–100 g) of
protein from each sample were loaded on 12% re-
solving polyacrylamide gels and then transferred to
Immobilon-P membrane (Millipore Corp., Marlbor-
ough, 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 (Amer-
sham Life Science Inc., Arlington Heights, IL) per
the manufacturer’s protocol.
Southern blot analysis. To determine the inde-
pendence of C-33A clonal sublines transfected with
pcDNA3.1/zeo+/FHIT or pcDNA3.1/zeo+, South-
ern 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 trans-
ferred to Zeta probe membrane (Bio-Rad Laborato-
ries, Inc., Hercules, CA). Hybridization to a
32
P-
labeled pcDNA3.1/FHIT probe was carried out in
Rapid Hyb Solution (Amersham Life Science Inc.),
as recommended by the manufacturer. The mem-
branes were exposed to XAR5 autoradiography film
(Kodak, Rochester, NY) for varying lengths of time
at −80 °C. Several clones revealing unique band pat-
terns were selected for further analysis.
Colony formation in soft agar. Dishes (35 mm)
were precoated with 1 mL of 0.6% agar/1× complete
medium (medium with FBS). Suspensions of 10
4
cells in 0.3% agar/1× 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 m in diameter was
counted with the aid of a microscope eyepiece con-
taining a grid.
Tumorigenicity in nude mice. Polyclonal popu-
lations 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 × 10
6
cells in 0.2-mL Hanks’ bal-
anced salt solution without phenol red (Life Tech-
nologies, Inc.). Cells transfected/transduced with
each construct were injected into five mice, which
were ear-tagged and followed individually through-
out 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 pe-
riods of time resulted in significant tumor necrosis.
Animals were killed with CO
2
, 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 por-
tions for further analyses. Representative samples of
each tumor were fixed in 10% buffered formalin,
routinely processed, and embedded in paraffin. Tis-
sue sections (5 m) 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 im-
munohistochemistry. Representative portions of xe-
nograft 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 ani-
mal studies were conducted in accordance with a
study protocol approved by the University of Michi-
gan’s Committee on Use and Care of Animals.
Immunofluorescent staining of Fhit protein.
Approximately 10
5
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 incu-
bated 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 incu-
bated 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 (Olym-
pus, Melville, NY).
Immunohistochemical staining of Fhit protein.
Tissue sections (5 m) 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 per-
oxidase 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 over-
Journal of the National Cancer Institute, Vol. 92, No. 4, February 16, 2000 REPORTS 339
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night at 4 °C. After washing in PBS, slides were
incubated with a biotinylated goat anti-rabbit sec-
ondary antibody for 30 minutes at room tempera-
ture. Antigen–antibody complexes were detected
with the avidin–biotin–peroxidase method by use of
diaminobenzidine as a chromogenic substrate (Vec-
tastain ABC-Immunostaining Kit; Vector Laborato-
ries, Inc., Burlingame, CA), as recommended by the
manufacturer. Tissue sections were lightly counter-
stained 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 involv-
ing colony formation in soft agar, the number of
colonies in each dish more than 100 m were
counted. Separate counts were recorded for colonies
100–200 m, 201–300 m, and greater than 300
m. The remainder of the initial group of 10
4
cells
were placed into the less than 100-m 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 m
between the FHIT and control groups. Each analysis
was performed separately by cell line. For the ex-
periments 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 ex-
periments 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. Sepa-
rate 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 poly-
clonal cell populations.
RESULTS
C-33A, C-4II, AGS, and H460 Cells
and Expression of Endogenous Fhit
Protein
We evaluated several cervical carci-
noma 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 pro-
tein 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 kera-
tinocytes (Fig. 1, A). Our detection of
full-length FHIT transcripts and Fhit pro-
tein in HeLa contrasts with other studies
in which Fhit protein was reported to be
undetectable (18). The different observa-
tions 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, form-
ing poorly differentiated squamous carci-
nomas 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 stud-
ies, the injection of up to1×10
7
AGS
gastric carcinoma cells failed to generate
tumors in nude mice during a 9-week sur-
Fig. 1. Immunoblot analysis of fragile histidine triad (Fhit) protein expression in selected cancer cell lines.
Panel A: Expression of endogenous Fhit protein in human primary foreskin keratinocytes (PFK) and in six
human cervical carcinoma-derived cell lines (C-4II, CaSki, HeLa, ME180, SiHa, and C-33A) (100 g total
protein was loaded in each lane). Panel B: Expression of Fhit in polyclonal populations of C-33A and C-4II
cervical carcinoma cells and H460 lung carcinoma cells generated by stable transduction with recombinant
retroviruses (30 g total protein loaded in each lane). Total protein was isolated from cells stably transduced
by FHIT recombinant retroviruses (F) or retroviruses lacking the FHIT insert (V). Fhit expression in
preinjection cells (C) and derivative tumor xenografts (X)isshown.Panel C: Fhit expression in polyclonal
(P) and clonal (CL) C33A cell populations generated by stable transfection with pcDNA3.1/zeo+ (V)and
pcDNA3.1/zeo+/FHIT (F) expression plasmids (amounts of total protein loaded are indicated). Fhit expres-
sion in preinjection cells from one control clone V(CL1) and three Fhit expressing clones F(CL1–3) is
compared with Fhit expression in representative derivative xenografts F(CL2) and F(CL3).
340 REPORTS Journal of the National Cancer Institute, Vol. 92, No. 4, February 16, 2000
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veillance period. AGS cells were, there-
fore, excluded from further studies of
Fhit’s ability to suppress their tumorigen-
ic growth in nude mice but included in
our analysis of Fhit’s effects on anchor-
age-independent growth.
Restoration of Fhit Expression in
C-33A, C-4II, and H460 Cells
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 pro-
tein of the expected size was easily de-
tected 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 in-
fected with a retrovirus lacking the FHIT
cDNA insert (Fig. 1, B, lanes 7–9). Ex-
pression 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 plas-
mid—three 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 alone—
representative 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 retrovi-
ruses was also confirmed by immunoflu-
orescence staining prior to further analy-
sis (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 obvi-
ous changes in cellular morphology or
growth kinetics were noted in cells ex-
pressing Fhit protein.
Restoration of Fhit Expression and
Tumorigenicity of Cervical or H460
Lung Carcinoma Cells
The parental cell lines were subcutane-
ously injected into nude mice to confirm
their tumorigenic growth properties. In-
jection of5×10
6
cells from the C-33A
and C-4II cervical cancer lines and the
H460 lung cancer line resulted in forma-
tion of palpable tumors after 2 weeks. Sta-
bly selected polyclonal populations of
each cell line infected with either recom-
binant 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 indepen-
dent, stably transfected C-33A clones
Fig. 2. Immunofluorescent and immunohistochemical staining of fragile histidine triad (Fhit) protein in
cancer cell lines and xenografts. Immunofluorescent staining of Fhit protein in polyclonal populations of A)
C-33A cervical carcinoma cells, B) C-4II cervical carcinoma cells, and C) H460 lung carcinoma cells stably
transduced by FHIT recombinant retroviruses. Immunohistochemical staining of Fhit protein in tumor
xenografts from cells transfected with the pcDNA3.1/zeo+/FHIT expression plasmid: D) C-33A clone 1
(original magnification ×100); E) C-33A clone 1 (original magnification ×400); from polyclonal populations
of cells infected with FHIT recombinant retroviruses: F) C-33A (original magnification ×100); G) C-33A
(original magnification ×400); H) C-4II (original magnification ×400); I) H460 (original magnification
×400); and from cells infected with retroviruses lacking the FHIT insert: J) C-33A (original magnification
×400); K) C-4II (original magnification ×400); and L) H460 (original magnification ×400).
Journal of the National Cancer Institute, Vol. 92, No. 4, February 16, 2000 REPORTS 341
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[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 poly-
clonal or clonal cell lines tested. The net
weights and volumes of tumor tissues ob-
tained immediately after excision from
euthanized mice are shown in Fig. 3,
A–D. There was no statistically signifi-
cant 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 con-
trol (P>.05; mixed models; Fig. 3, A and
B). Similarly, there was no statistically
significant difference in the weight or vol-
ume of tumors formed by clonal popula-
tions 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 pa-
rental and vector-only transduced C-4II
cells were rather small, with in vivo re-
gression observed starting 2 weeks after
injection. Tumor formation by C-4II cells
was documented by microscopic exami-
nation of xenograft tissues, and no differ-
ences were noted between Fhit-positive
and the Fhit-negative cells. Tumor xeno-
grafts formed by Fhit-expressing C-33A,
C-4II, and H460 cells were shown to re-
tain strong Fhit protein expression by
both immunoblot and immunohistochem-
ical analyses. Fig. 1, B (lanes 2, 4, and 6),
shows retention of Fhit expression in xe-
nografts derived from polyclonal popula-
tions of cells transduced by recombinant
retroviruses. Fig. 1, C (lanes 7 and 8),
shows retention of Fhit expression in xe-
nografts derived from clonal populations
of C-33A cells stably transfected with the
pcDNA3.1/zeo+/FHIT plasmid. Immuno-
histochemical detection of Fhit protein
expression in representative tumor xeno-
grafts 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 trans-
fected with pcDNA3.1/zeo+/FHIT. Pan-
els F and G show Fhit expression in a
xenograft derived from polyclonal C-33A
cells transduced with the pPGS−CMV−
CITE−Neo(+)/FHIT recombinant retrovi-
rus. 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 xeno-
grafts, with the xenografts retaining a sub-
stantial number of cells strongly express-
ing Fhit. For comparison purposes, the
absence of Fhit expression in representa-
tive xenografts formed by polyclonal
populations of C-33A, C-4II, and H460
cells retrovirally transduced with vector
alone is shown in panels J–L.
Restoration of Fhit Expression and
Colony Formation in Soft Agar
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 express-
ing Fhit versus vector control (P>.05).
However, C-33A and H460 cells express-
ing 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.
DISCUSSION
Tumor suppressor genes are perhaps
most clearly distinguished by their bialle-
lic inactivation in cancer as a result of
deletions, point mutations, or epigenetic
modifications such as promoter methyl-
ation. In cervical carcinomas, inactivation
of tumor suppressor gene products can
also occur through interactions between
the HPV-transforming proteins E6 and E7
and the cellular proteins p53 and pRB,
respectively. Tumor suppressor genes can
also be defined functionally as genes
whose protein products inhibit the tumor-
igenic or metastatic properties of cells.
Because FHIT does not entirely conform
to the classical functional and genetic cri-
teria defining tumor suppressor genes, it
has been difficult to definitively prove or
exclude it as a bona fide tumor suppres-
sor. Our studies demonstrate that restora-
tion of Fhit expression fails to suppress
anchorage-independent growth or tumori-
genicity of two cervical carcinoma cell
lines in a xenograft model. Otterson et al.
(18) similarly found that Fhit is unable to
suppress the tumorigenic properties of
HeLa, another cervical cancer-derived
cell line.
Studies of Fhit’s ability to suppress tu-
morigenicity 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 Ot-
terson et al. have suggested, Fhit exerts its
Fig. 3. Weight and volume of tumor xenografts. Weight (A) and volume (B) volume of tumor xenografts
from polyclonal populations of C-33A, C-4II, and H460 cells transduced by fragile histidine triad (FHIT)
recombinant versus control retroviruses. Weight (C) and volume (D) volume of tumor xenografts derived
from clonal populations of C-33A cells transfected with pcDNA3.1/zeo+/FHIT (C-33A F1–3) or pcDNA3.1/
zeo+ vector alone (C-33A V1–3).
342 REPORTS Journal of the National Cancer Institute, Vol. 92, No. 4, February 16, 2000
at University of Michigan on January 23, 2012http://jnci.oxfordjournals.org/Downloaded from
tumor-suppressive effect in a cell lineage-
specific manner (18), Fhit may well sup-
press tumorigenicity of cell lines derived
from lung, kidney, or digestive tract can-
cers but not those derived from cervical
cancers. However, this explanation can-
not account for the different results ob-
tained by three independent groups study-
ing H460 lung carcinoma cells. Genetic
heterogeneity of H460 sublines utilized in
different laboratories may be, in part, re-
sponsible for the different outcomes be-
cause genetic variation at the FHIT locus
has been described in other cancer cell
sublines such as HeLa (27,30). The con-
tradictory results may also reflect differ-
ences in the level and degree of hetero-
geneity of exogenous Fhit expression ob-
tained 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 ex-
press exogenous Fhit protein, even when
clonal sublines of cells were isolated.
Other investigators may have achieved
more uniformly elevated levels of Fhit ex-
pression in the cells that they tested. How-
ever, if heterogeneity of Fhit expression is
solely responsible for the different out-
comes, 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 problema-
tic results. Notably, we found compar-
able levels of Fhit expression in clonal
preinjection cells and their derivative xe-
nografts, regardless of whether the cells
initially expressed high [e.g., C-33A-
F(CL2)] versus low [C-33A-F(CL3)] lev-
els of Fhit protein (Fig. 1, C).
Although our study fails to provide ad-
ditional 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 ex-
pression in cervical cancer precursor le-
sions, 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 nu-
merous other genetic alterations may not
be sufficient for demonstrably affecting
their growth or tumorigenic properties.
Clearly, additional studies will be re-
quired to fully understand the role of
FHIT in human cancer.
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Cell line† 101–200
m201–300
m>300
mPlinear
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NOTES
Supported by Public Health Service grants
CA64466 and CA81587 from the National Cancer
Institute, National Institutes of Health, Department
of Health and Human Services.
We thank Eric R. Fearon for his helpful discus-
sions and review of this manuscript.
Manuscript received June 10, 1999; revised No-
vember 18, 1999; accepted December 6, 1999.
344 REPORTS Journal of the National Cancer Institute, Vol. 92, No. 4, February 16, 2000
at University of Michigan on January 23, 2012http://jnci.oxfordjournals.org/Downloaded from
... FHIT loss on cancer cells favors tumor progression and metastasization and is associated with a poor prognosis [22][23][24]. This gene has been found to inhibit the proliferation of tumor cells, induce their apoptosis, and increase invasion and their susceptibility to chemotherapeutic agents [25,26]; however, it has been also reported that FHIT does not act as a direct tumor suppressor gene [27][28][29]. ...
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Allelic losses involving chromosome 3p are frequently observed in cervical cancers. Deletion mapping studies of primary cervical carcinomas have localized common regions of deletion to 3p14.2 and 3p21. The candidate tumor suppressor gene FHIT has been mapped to 3p14.2, and previous studies have demonstrated reduced or aberrant FHIT transcripts and reduced or absent Fhit protein expression in a large percentage of cervical cancer-derived cell lines and primary cervical carcinomas. To expand these observations to preinvasive cervical epithelial lesions and to determine whether loss of Fhit protein expression might be associated with tumor progression, immunohistochemical methods were used to examine Fhit expression in 95 invasive cervical carcinomas, 33 high-grade squamous intraepithelial lesions (HSILs) associated with concurrent invasive cancer, 38 HSILs unassociated with invasive cancer, 24 low-grade squamous intraepithelial lesions, and 22 normal cervix samples. All normal cervical epithelia and low-grade squamous intraepithelial lesions exhibited diffuse cytoplasmic immunostaining of moderate to strong intensity. Fhit protein expression was markedly reduced or absent in 67 of 95 (71%) invasive cancers, 17 of 33 (52%) HSILs associated with invasive cancer, and 8 of 38 (21%) HSILs without associated invasive cancer. The results confirm that Fhit protein expression is reduced or absent in the majority of cervical carcinomas and suggest that loss of Fhit expression often accompanies cervical tumor progression. Moreover, absent or reduced Fhit protein is observed at a significantly higher frequency in HSILs associated with progression to invasive cancer than in HSILs with unknown risk for progression (P = 0.012). These findings suggest that loss of Fhit expression in HSILs could serve as a useful marker of high-grade preinvasive lesions that have an increased likelihood of progression to invasive carcinoma.
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To determine whether the FHIT gene at 3p14.2 is altered in head and neck squamous cell carcinomas (HNSCC), we examined 26 HNSCC cell lines for deletions within the FHIT locus by Southern analysis, for allelic losses of specific exons FHIT by fluorescence in situ hybridization (FISH) and for integrity of FHIT transcripts. Three cell lines exhibited homozygous deletions within the FHIT gene, 55% (15/25) showed the presence of aberrant transcripts, and 65% (13/20) showed the presence of multiple cell populations with losses of different portions of FHIT alleles by FISH of FHIT genomic clones to interphase nuclei. When the data obtained by FISH and by reverse transcriptase-PCR analyses are combined, 22 of 26 cell lines showed alterations of at least one allele of the FHIT gene. Our data indicate that the FHIT gene is disrupted in HNSCCs and hence, loss of FHIT function may be important in the development and/or progression of head and neck cancers.
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A 200-300 kb region of chromosome 3p14.2, including the fragile site locus FRA3B, is homozygously deleted in multiple tumor-derived cell lines. Exon amplification from cosmids covering this deleted region allowed identification of the human FHIT gene, a member of ther histidine triad gene family, which encodes a protein with 69% similarity to an S. pombe enzyme, diadenosine 5', 5''' P1, P4-tetraphosphate asymmetrical hydrolase. The FHIT locus is composed of ten exons distributed over at least 500 kb, with three 5' untranslated exons centromeric to the renal carcinoma-associated 3p14.2 breakpoint, the remaining exons telomeric to this translocation breakpoint, and exon 5 within the homozygously deleted fragile region. Aberrant transcripts of the FHIT locus were found in approximately 50% of esophageal, stomach, and colon carcinomas.