Clinical Cancer Research Joint Metastasis Research Society-AACR Conference on Metastasis Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Holschneider, C. H.
Right arrow Articles by Karlan, B. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Holschneider, C. H.
Right arrow Articles by Karlan, B. Y.
Clinical Cancer Research Vol. 11, 5756-5763, August 15, 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

The Fragile Histidine Triad Gene: A Molecular Link Between Cigarette Smoking and Cervical Cancer

Christine H. Holschneider1,2,3, Rae Lynn Baldwin2, Kiran Tumber2, Chisa Aoyama4 and Beth Y. Karlan1,2

Authors' Affiliations: 1 Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, 2 Women's Cancer Research Institute, Cedars-Sinai Medical Center, Los Angeles; Departments of 3 Obstetrics and Gynecology and 4 Pathology, Olive View-UCLA Medical Center, Sylmar, California

Requests for reprints: Christine H. Holschneider, Gynecologic Oncology, Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, 10833 Le Conte Avenue, CHS 24-127, Los Angeles, CA 90095-1740. E-mail: Christine.Holschneider{at}cshs.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Smoking is an epidemiologic risk factor for cervical cancer. The fragile histidine triad (FHIT) gene is a tumor suppressor gene that is altered in 80% of tobacco-associated lung cancers. We hypothesized that reduced FHIT protein expression, homozygous deletions (HD) or hemizygous deletions (HemiD) and microsatellite alterations (MA) at the FHIT/FRA3B locus occur more commonly in cervical cancers of smokers than nonsmokers.

Experimental Design: Archival tissues of 58 patients with stage IA1 to IB2 squamous cell carcinoma of the cervix were identified. FHIT protein expression was studied with immunohistochemistry. Laser capture microdissection was used to isolate tumor and normal DNA. HD/HemiD of FHIT exons 4 and 5 were analyzed by monoplex real-time PCR. MA at FHIT/FRA3B were studied with multiplex nested PCR with three fluorescently labeled microsatellite markers (D3S1300, D3S1312, and D3S1480).

Results: Eighteen of 26 tumors from smokers (69%) and 13 of 32 nonsmokers (41%; P < 0.05) showed loss of FHIT protein expression. Thirty-seven stage IB tumors yielded sufficient DNA for analyses. HD or HemiD of both exons tested occurred in 8 of 17 smokers (47%) and 2 of 20 nonsmokers (10%; P < 0.05). MA at more than two sites were found in 11 of 17 tumors of smokers (65%) and 6 of 20 nonsmokers (30%; P < 0.05). Mean composite genomic FHIT alteration scores were significantly higher for tumors of smokers versus nonsmokers (0.67 versus 0.40; P < 0.02).

Conclusion: Loss of FHIT expression, HD, HemiD, and MA at the FHIT/FRA3B locus occur significantly more commonly in cervical cancers of smokers. These findings suggest that the tumor suppressor gene FHIT may represent a molecular target in cigarette smoking–associated cervical carcinogenesis.

The human papillomavirus (HPV) is detected in 99.7% of all squamous cell carcinomas of the uterine cervix (1) and is thought to be essential for cervical carcinogenesis. However, reported annual HPV infection rates are ~1,000 times higher than the annual incidence of cervical cancer and at least 90% of HPV infections resolve spontaneously (2–4). Thus, HPV seems necessary, but by itself is not sufficient to cause cervical cancer. Other carcinogenic cofactors become important, one of which is cigarette smoking. Epidemiologic studies implicate cigarette smoking as an independent risk factor for the development of cervical cancer, with odds ratios ranging from 1.8 to 4.3 (5, 6). Cigarette smoke constituents, such as nicotine, cotinine, benzo(a)pyrene, and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone concentrate in the mucous of the uterine cervix (7–10). This led us to postulate that cigarette smoking–associated cervical cancer may share certain pathogenetic features with other tobacco-associated malignancies.

Studies of lung cancers and premalignant pulmonary lesions have shown a strong association between cigarette smoking and structural or epigenetic alterations of the fragile histidine triad gene (FHIT; refs. 11–13), a tumor suppressor gene (14) located on chromosome 3p14.2 (15). Loss of heterozygosity (LOH) at FHIT is found in 80% of lung cancers of smokers compared with 22% of lung cancers of nonsmokers (11). In cervical neoplasia, allelic deletions involving the short arm of chromosome 3 (3p13-21.1) have been found in 40% to 70% of cancers and in up to 61% of intraepithelial neoplasias (16–18), with LOH at 3p14.2 occurring in 56% of cancers and 20% of intraepithelial neoplasias (17). To date, it remains unknown whether an association between cigarette smoking and FHIT alterations also exists for cervical cancer.

With this information as background, we postulated that the FHIT gene might be a candidate target gene involved in cigarette smoking–associated carcinogenesis in the uterine cervix. We hypothesized that reduced FHIT protein expression, homozygous deletions (HD) and/or hemizygous deletions (HemiD), as well as microsatellite alterations (MA) at the FHIT/FRA3B locus occur more commonly in cervical cancers of smokers than nonsmokers. To test this hypothesis, we studied archival tissues from 58 patients (26 smokers and 32 nonsmokers) with squamous cell carcinoma of the uterine cervix using immunohistochemistry of tumor and adjacent dysplastic and nondysplastic squamous epithelium and PCR-based analysis of tumor and matched nontumor control DNA.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and archival tissue samples. Approval for this historical cohort study was obtained from the Institutional Review Boards. Patients who underwent primary surgical therapy between January 1990 and June 2003 for stage IA1 to IB2 squamous cell carcinoma of the uterine cervix with no evidence of lymph node metastases were identified through surgical logs and institutional tumor registries. Data on patient age, race, smoking habits, and tumor grade were extracted from the medical record. Patients were included in the study if smoking status (current smoker or never smoker) was clearly documented in at least two different sites in the hospital chart. Ex-smokers were excluded. Identified cases were reviewed by a gynecologic pathologist for identification of sites for immunohistochemistry and DNA isolation by laser capture microdissection. Only cases for which cancer and adjacent nondysplastic squamous epithelium were available were included. Formalin-fixed, paraffin-embedded tissues were available for 58 identified patients (26 smokers and 32 nonsmokers). There were 21 stage IA and 37 stage IB squamous cell carcinomas. Immunohistochemistry analyses were done on all 58 cases. Laser capture microdissection and DNA analyses were done only on the 37 stage IB cancers, which had sufficient numbers of tumor cells available. From each specimen block, contiguous sections were used for immunohistochemistry and laser capture microdissection.

Immunohistochemical analysis of FHIT protein expression. Slide-mounted tissue sections were deparaffinized in three 10-minute xylene washes and rehydrated through a series of graded alcohols. Antigen retrieval was done by incubating tissue sections in sodium citrate buffer (10 mmol/L, pH 6.0) at 95°C for 20 minutes. Endogenous peroxidase was inactivated by treatment with 3% hydrogen peroxide in methanol. Slides were incubated in 2% bovine serum albumin blocking solution, followed by incubation with a polyclonal rabbit anti-human FHIT antibody at a 1:100 dilution (Zymed Laboratories Inc., San Francisco, CA) for 1 hour at room temperature. Slides were treated with biotinylated secondary antibodies and antibody binding sites were visualized using 3,3'-diaminobenzidine (DAKO Corp., Carpinteria, CA). Slides were lightly counterstained with H&E. Immunostains of tumor, tumor adjacent dysplastic, and nondysplastic squamous epithelium were scored for intensity of staining present in >50% of the tissue of interest using a four-tiered scale (0, absent; 1, weak; 2, moderate; 3, intense staining). Changes in FHIT protein expression in tumor and dysplasia was scored in each case relative to the nondysplastic tumor adjacent squamous epithelium as an internal control. All slides were scored independently by two examiners. Inter-examiner agreement for the immunohistochemistry analysis was 89%. The six cases with disagreement on the initial scoring were reviewed by both examiners together and a consensus score was calculated and used for the statistical analysis.

Laser capture microdissection and DNA isolation. To isolate tumor cells, laser capture microdissection (Arcturus Engineering, Mountain View, CA) was done on H&E-stained tissue sections. Lymph nodes with no evidence of metastases were used for normal control DNA. DNA was extracted from cells captured using the Pico Pure DNA Extraction Kit (Arcturus Engineering). Incubation was carried out for 96 hours at 65°C in a volume of 50 µL extraction buffer containing 50 µg proteinase K. At 24-hour intervals, an additional 10 µg of proteinase K was added to each sample. Proteinase K was inactivated at 95°C for 10 minutes. DNA was purified using MicroSpin G-25 Columns (Amersham Biosciences, Buckinghamshire, England). DNA yield was spectrophotometrically quantified. After further dilution, this DNA eluate was used directly as a template for the PCR reactions.

Homozygous deletions of FHIT exons 4 and 5. Real-time quantitative PCR amplification of FHIT exons 4 and 5 was done with previously published oligonucleotide primers (19) using the iCycler (Bio-Rad, Hercules, CA) and SYBR green supermix (Bio-Rad). Reactions were carried out in a total volume of 25 µL using 150 ng template DNA. PCR conditions were as follows: an initial denaturation at 95°C for 3 minutes, followed by 47 cycles of 95°C for 10 seconds and 60.4°C (exon 4) or 63.5°C (exon 5) for 45 seconds. After amplification, melt curve analysis was done to verify amplification specificity by heating the reaction mixture from 55°C to 95°C at the rate of 0.05 °C/s. Data analysis was carried out using the iCycler iQ Optical Systems software version 3.0a (Bio-Rad). FHIT exons 4 and 5 were chosen for study as they flank intron 4, the most active area of the common fragile site FRA3B (refs. 20–24; Fig. 1). A standard curve was formulated for FHIT exons 4 and 5 and glyceraldehyde-3-phosphate dehydrogenase GAPDH using serum leukocyte DNA dilution series over 4 logs. Only quantitative PCR reactions whose standard curves had correlation coefficients ≥0.98 were used. Using each sample's Ct value against the respective standard curves, the starting DNA quantity was calculated and subsequently normalized to the respective sample's DNA content as determined by the amplification of the housekeeping gene, GAPDH yielding the gene dosage ratio (GDR). All PCRs were repeated in triplicate and starting DNA quantities averaged for each sample. Negative water and positive serum leukocyte DNA controls were run with each PCR reaction. In addition, all PCR products were separated by 3% agarose gel electrophoresis and visualized with ethidium bromide staining. Only samples for which the gel showed an amplification product of expected size and the melt curve was within the expected temperature range were included for analysis. HD was scored when a sample had a GDR ≤0.36. This allows for the presence of some contaminating normal cells, which are invariably present even in tumor cells separated by laser capture microdissection. A GDR between 0.36 and 0.63 was interpreted as HemiD, i.e., under-representation of the test gene FHIT relative to the reference gene GAPDH. A GDR of ≥0.63 was interpreted as having no deletion of the target gene.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 1. The FHIT gene, which encodes a 1.1 kb mRNA, spans 1.8 Mb. It is encompassed by FRA3B, the most active of the common fragile sites, which spans a total of 4 Mb. Within FRA3B, the characteristic chromosome gaps can occur anywhere, but the majority occur in introns 4 and 5, surrounding the first protein coding exon (exon 5). FRA3B is a known HPV integration site (20–24).

 
Microsatellite alterations at FHIT/FRA3B. Paired normal and tumor DNA samples were amplified by hot-start multiplex nested PCR using nonlabeled locus-specific flanking primer pairs for the three nested fluorescently labeled microsatellite markers (D3S1300-FAM, D3S1312-HEX, and D3S1480-FAM). These markers were chosen for their location within FHIT/FRA3B and close proximity to previously identified HPV integration sites (refs. 20–24; Fig. 1). The multiplex flanking PCR was done with an initial denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 61°C for 30 seconds, and extension at 72°C for 1.5 minutes, with a final extension at 72°C for 7 minutes. To reduce carryover of flanking primers into the nested PCR reaction, PCR products were purified using the GenElute PCR clean up kit (Sigma, St. Louis, MO). Nested PCR conditions were as follows: an initial denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 15 seconds, annealing at 54.2°C (D3S1300) or 56.2°C (D3S1312) or 53.0°C (D3S1480) for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 7 minutes. PCR products were analyzed on a DNA sequencer (ABI 3700) using ABI prism genotyper 2.5 for analysis (Global Medical Instrumentation, Inc., Ramsey, MN). Areas under the curve (AUC) were calculated for each allele and AUC ratios between the short and long allele were calculated for all normal and tumor samples of informative cases. LOH was defined as an AUC ratio differential >70% between the tumor alleles and the corresponding normal alleles. Intragenic microsatellite instability was defined as gain of a novel length allele in the tumor DNA. LOH and intragenic microsatellite instability were collectively referred to as microsatellite alterations. All MA data were confirmed in triplicate. The range of possible microsatellite findings for D3S1300 is illustrated in Fig. 2.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 2. Four possible microsatellite findings using D3S1300 as an example: heterozygous, no loss (A); homozygous, noninformative (B); microsatellite instability, MSI (C); loss of heterozygosity, LOH (D). The two numbers under each allele peak designate allele length and AUC.

 
Data analysis and statistics. Statistical analyses were done using the JMP Statistical Discovery Software (SAS Institute, Inc., Cary, NC). Primary outcome measures were (a) FHIT protein expression by immunohistochemistry, (b) HD/HemiD of FHIT exons 4 and 5, and (c) MA at the FHIT/FRA3B locus in the cervical cancers as compared between smokers and nonsmokers. These two patient groups were further compared with regard to patient age, race, and tumor grade. The Wilcoxon ranked sums test and Kruskal-Wallis test were used for nonparametric testing of scores or continuous variables as indicated. Fisher's exact test was used for categorical variables.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemistry showed progressive loss of FHIT expression from tumor-adjacent nondysplastic squamous epithelium (mean score, 1.98; 95% confidence intervals, 1.79-2.17) to adjacent severe dysplasia (mean score, 1.61; 95% confidence intervals, 1.36-1.86) to invasive cancer (mean score, 1.36; 95% confidence intervals, 1.12-1.60; P < 0.001; Fig. 3). Lost or reduced FHIT protein expression relative to adjacent nondysplastic squamous epithelium was found in 31 tumors (53%) with no significant difference between very early (stage IA) and more advanced (stage IB) invasive cancers (48% versus 57%). FHIT protein expression was unaltered in 27 tumors (47%), again with no significant difference between very early (stage IA) and more advanced (stage IB) invasive cancers (52% versus 43%; Fig. 4). Lost or reduced FHIT expression was significantly (P < 0.01) more common in tumors of smokers (18 of 26; 69%) than those of nonsmokers (13 of 32; 41%). In tumor-adjacent severe dysplasia, reduced FHIT expression was noted in 7 of 17 (41%) of smokers and 7 of 20 (35%) of nonsmokers. Of note, specimens from 9 smokers and 12 nonsmokers did not have any tumor-adjacent severe dysplasia available for analysis. There was no significant difference between the two groups for patient age, race, and tumor grade (Table 1).



View larger version (72K):
[in this window]
[in a new window]
  		Fig. 3. Immunohistochemistry for FHIT with H&E counterstain, x120. Progressive loss of FHIT protein expression with progressive cervical neoplasia in the same patient: moderate (2+) FHIT staining in nondysplastic squamous epithelium (A), weak (1+) staining in severe dysplasia (B), and absent FHIT staining in invasive squamous cell carcinoma (C). These changes parallel the significant differences obtained in average immunohistochemistry scores from all specimens for tumor-adjacent nondysplastic epithelium, severe dysplasia, and cancer (P < 0.001).

 


View larger version (96K):
[in this window]
[in a new window]
  		Fig. 4. Immunohistochemistry for FHIT with H&E counterstain, x24.5. FHIT protein expression in tumor versus adjacent nondysplastic epithelium as internal control. Tumor and adjacent epithelium with significantly reduced staining for FHIT protein in tumor (A) compared with a tumor and adjacent epithelium with intense staining of tumor (B). C, cancer; E, epithelium.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Comparison of clinical and molecular characteristics of the two study groups, smokers and nonsmokers

 
The 37 stage IB tumors were studied for evidence of FHIT gene alterations by performing PCR-based analyses for HD/HemiD and MA (Tables 1 and 2). HD or HemiD of both exons tested occurred in 8 of 17 smokers (47%) and 2 of 20 nonsmokers (10%; P < 0.05). MA at two or more sites was found in 11 of 17 tumors of smokers (65%) and 6 of 20 nonsmokers (30%; P < 0.05; Table 1). These differences for HD, HemiD, and MA between smokers and nonsmokers were still significant if the statistical analysis was limited to the evaluable and informative cases only. Composite genomic FHIT alteration scores were then calculated by determining the proportion of sites with HD, HemiD, and/or MA over the number of informative and evaluable sites for each case. Median composite genomic FHIT alteration scores were significantly higher for tumors of smokers versus those of nonsmokers (0.67 versus 0.40; P < 0.02; Table 2). The observed median difference in scores was 0.27 (95% confidence interval, 0.03-0.41).


View this table:
[in this window]
[in a new window]
  		Table 2. Summary of PCR results for microsatellite markers D3S1300, D3S1312, and D3S1480 and FHIT exons 4 and 5 in 37 tumors (17 smokers and 20 nonsmokers)

 
The correlation between FHIT immunohistochemistry and genomic FHIT alterations as determined by HD/HemiD and/or MA was evaluated. There was excellent correlation between HD or HemiD at FHIT exons 4 and 5 and FHIT protein expression in the sense that all cases with HD and/or HemiD at both FHIT exons 4 and 5 had reduced or lost FHIT protein expression. Reversely, of the cases with reduced FHIT protein expression, only one had no evidence of genomic FHIT alteration at any of the three microsatellite markers or two FHIT exons tested.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Cervical cancer likely results from the accumulation of a series of genetic and epigenetic events such as HPV integration, activation of oncogenes, or inactivation of tumor suppressor genes. One such gene found to be affected in cervical cancer is the tumor suppressor gene FHIT (17, 25–27), which was cloned by Ohta et al. in 1996 (15).

FHIT is one of the most frequently altered genes in a wide range of solid tumors (14). After its initial discovery, there was substantial controversy whether FHIT is a bona fide tumor suppressor gene involved in the carcinogenesis process or whether its frequent alterations are merely a bystander effect due to its location at a well-known genetically fragile site, FRA3B. Over the past 8 years, strong evidence has developed to support the postulation that FHIT is a tumor suppressor gene. Transfection of FHIT DNA reduces the tumorigenicity of different cancer cell lines in nude mice (28, 29). FHIT overexpression induces apoptosis (29–31). FHIT knock-out mice show increased susceptibility to spontaneous and induced tumors, and oral gene therapy using adeno and adeno-associated FHIT viruses prevents and reverses forestomach tumors in FHIT mutant mice (32–34). Despite the evidence indicating that FHIT functions as a tumor suppressor, the signaling pathway through which FHIT induces apoptosis and inhibits growth of cancer cells is poorly understood.

It has been postulated by Huebner and colleagues that the FHIT/FRA3B locus is particularly susceptible to the effects of environmental carcinogens (35). As shown by molecular epidemiologic data on lung cancer, one such carcinogen is cigarette smoke. Preinvasive and invasive pulmonary neoplasia shows significant FHIT alterations in a large proportion of lesions (36). Reduction or complete loss of FHIT expression by immunohistochemistry is seen in about 30% to 70% of non–small cell lung cancer and in about 20% of bronchial biopsies of chronic smokers without evidence of lung cancer (37).

In our current study, we are the first to document the link between cigarette smoking and FHIT alterations in cervical cancer, with significantly more tumors of smokers having shown loss of FHIT protein expression (69% versus 41%). This was paralleled by a difference of comparable magnitude in genomic FHIT alterations between tumors of smokers and nonsmokers. These findings suggest that the tumor suppressor gene FHIT may represent a target in cigarette smoking–associated cervical carcinogenesis. These observation support our hypothesis that FHIT, located at the fragile site FRA3B, is a potential genetic link between cigarette smoking and cervical cancer.

Contrary to other tumor suppressor genes, inactivating point mutations are rarely observed for FHIT and chromosomal rearrangements and deletions are the predominant genetic alterations (35). In addition, studies have shown that aberrant methylation of FHIT causes promoter silencing (38) and an increased frequency of aberrant FHIT methylation was shown in high-grade cervical dysplasia and cancer (39). Furthermore, in lung cancer aberrant methylation of the FHIT gene has been found to be associated with cigarette smoking (13). What remains unclear is the causative impact of these genetic and epigenetic alterations and whether they compliment each other or act independently during carcinogenesis in general, and in the smoking-associated neoplastic process in particular.

Homozygous deletion is a commonly observed phenomenon at the FHIT locus in a variety of tumors (40). Such apparent HDs may also result from the overlap of two independent deletions on each allele (41). Despite its many advantages, quantitative real-time PCR is associated with some technical limitations for the detection of homozygous and hemizygous deletions, including the concern for possible misclassification especially in the setting of contaminating normal DNA, gross tumor aneuploidy, or severe heterogeneity of subclones of tumor cells. Theoretically, normal tissue in which each cell has two copies of each gene has a GDR of 1 between the gene of interest and the reference gene. By the same principle, a euploid tumor with a homozygous deletion of the gene of interest has a GDR of 0, and a tumor with HemiD has a GDR of 0.5. However, ex vivo tumor tissue samples are, even after microdissection, likely contaminated with some normal DNA, may be aneuploid, or contain heterogeneous subclones. To account for these possibilities, as well as for some variation within the assay, we used a scoring system with GDRs of 0.36 and 0.63 as cutoffs for HD and HemiD, as previously described (42). Similarly, LOH was assigned when the AUC ratio of the tumor was reduced by at least 70% compared with the AUC ratio of the constitutional DNA, a cutoff which is in keeping with the literature on LOH analyses after microdissection (43, 44) and corresponds to the cutoff levels used for HD and HemiD.

A direct correlation between FRA3B fragile site expression and cigarette smoking has recently been shown in peripheral blood lymphocytes by Stein et al. (45). This association becomes particularly intriguing given recent data that suggest common fragile sites as preferential targets for HPV integration (23, 46), with FRA3B being a repeatedly identified site (21, 23, 47). HPV integration in turn is a pivotal event in the transition to invasive cervical cancer (48). Increased genetic instability has been reported to occur after integration of exogenous DNA at FRA3B (49) and FHIT alterations associated with cervical cancer seem to cluster in the most active portion of the fragile site FRA3B, which spans FHIT introns 4 and 5 (25).

The observed occurrence of HDs, HemiDs, and MAs, in particular in cancers of cigarette smokers in our study, may be evidence of such increased genetic instability in tumors exposed to cigarette smoke carcinogens and oncogenic HPV. To investigate this question further, functional in vitro experiments will allow us to assess the impact of acute and long-term exposure to cigarette smoke carcinogens on HPV and the expression of FHIT/FRA3B and other fragile sites.


   Acknowledgments
 
The authors thank Dr. Jeffrey Gornbein, Department of Biostatistics, David Geffen School of Medicine at UCLA, for assistance with the statistical analysis; Kent Taylor, PhD, Genotyping Core, Cedars-Sinai Medical Center, for assistance with the genotyping analysis; and Dr. Dan Scoles, Women's Cancer Research Institute, Cedars-Sinai Medical Center, for critical review of the manuscript.


   Footnotes
 
Grant support: Reproductive Scientist Development Program through NIH grant #5K12HD00849 and the American Board of Obstetrics and Gynecology and through the Burroughs Wellcome Fund (C.H. Holschneider), the Cedars-Sinai Women's Cancer Research Institute (B.Y. Karlan) and the Cedars-Sinai General Clinical Research Center, grant #MO1-RR00425.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/ 2/05; revised 4/26/05; accepted 5/20/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12–9.[CrossRef][Medline]
  2. Ho GY, Bierman R, Beardsley L, Chang CJ, Burk RD. Natural history of cervicovaginal papillomavirus infection in young women. N Engl J Med 1998;338:423–8.[Abstract/Free Full Text]
  3. Richardson H, Kelsall G, Tellier P, et al. The natural history of type-specific human papillomavirus infections in female university students. Cancer Epidemiol Biomarkers Prev 2003;12:485–90.[Abstract/Free Full Text]
  4. American Cancer Society. Cancer facts and figures 2004. Available at: http://www.cancer.org/downloads/STT/CAFF_finalPWSecured.pdf. Retrieved December 21, 2004.
  5. Winckelstein W, Jr. Smoking and cervical cancer—current status: a review. Am J Epidemiol 1990;131:945–57.[Free Full Text]
  6. Haverkos HW, Soon G, Steckley SL, Pickworth W. Cigarette smoking and cervical cancer: Part I. A meta-analysis. Biomed Pharmacother 2003;57:67–77.[CrossRef][Medline]
  7. Hellberg D, Nilsson S, Haley NJ, Hoffman D, Wynder E. Smoking and cervical intraepithelial neoplasia: nicotine and cotinine in serum and cervical mucus in smokers and nonsmokers. Am J Obstet Gynecol 1988;158:910–3.[Medline]
  8. Prokopczyk B, Cox JE, Hoffmann D, Waggoner SE. Identification of tobacco-specific carcinogen in the cervical mucus of smokers and nonsmokers. J Natl Cancer Inst 1997;89:868–73.[Abstract/Free Full Text]
  9. Sasson IM, Haley NJ, Hoffmann D, Wynder EL, Hellberg D, Nilsson S. Cigarette smoking and neoplasia of the uterine cervix: Smoke constituents in cervical mucus. N Engl J Med 1985;312:315–6.
  10. Melikian AA, Sun P, Prokopczyk B, et al. Identification of benzo[a]pyrene metabolites in cervical mucous and DNA adducts in cervical tissue in humans by gas chromatography-mass spectrometry. Cancer Lett 1999;146:127–34.[CrossRef][Medline]
  11. Sozzi G, Sard L, De Gregorio L, et al. Association between cigarette smoking and FHIT gene alterations in lung cancer. Cancer Res 1997;57:2121–3.[Abstract/Free Full Text]
  12. Nelson HH, Wiencke JK, Gunn L, Wain JC, Christiani DC, Kelsey KT. Chromosome 3p14 alterations in lung cancer: Evidence that FHIT exon deletion is a target of tobacco carcinogens and asbestos. Cancer Res 1998;58:1804–7.[Abstract/Free Full Text]
  13. Kim JS, Kim H, Shim YM, Han J, Park J, Kim DH. Aberrant methylation of the FHIT gene in chronic smokers with early stage squamous cell carcinoma of the lung. Carcinogenesis 2004;25:2165–71.[Abstract/Free Full Text]
  14. Huebner K, Croce CM. Cancer and the FRA3B/FHIT fragile locus: It's a HIT. Br J Cancer 2003;88:1501–6.[CrossRef][Medline]
  15. Ohta M, Inoue H, Cotticelli MG, et al. The FHIT gene, spanning the chromosome 3p14.2 fragile site and renal carcinoma-associated t(3;8) breakpoint, is abnormal in digestive tract cancers. Cell 1996;84:587–97.[CrossRef][Medline]
  16. Larson AA, Kern S, Curtiss S, Gordon R, Cavenee WK, Hampton GM. High resolution analysis of chromosome 3p alterations in cervical carcinoma. Cancer Res 1997;57:4082–90.[Abstract/Free Full Text]
  17. Wistuba II, Montellano FD, Milchgrub S, et al. Deletions of chromosome 3p are frequent and early events in the pathogenesis of uterine cervical carcinoma. Cancer Res 1997;57:3154–8.[Abstract/Free Full Text]
  18. Kersemaekers AM, Hermans J, Fleuren GJ, van de Vijver MJ. Loss of heterozygosity for defined regions on chromosomes 3, 11 and 17 in carcinomas of the uterine cervix. Br J Cancer 1998;77:192–200.[Medline]
  19. Fong KM, Biesterveld EJ, Virmani A, et al. FHIT and FRA3B 3p14.2 allele loss are common in lung cancer and preneoplastic bronchial lesions and are associated with cancer-related FHIT cDNA splicing aberrations. Cancer Res 1997;57:2256–67.[Abstract/Free Full Text]
  20. UCSC Genome Bioinformatics. UCSC Human Gene Sorter: FHIT on Human April 2003 Assembly. Available at: http://www.genome.ucsc.edu. Retrieved August 18, 2003 and December 29, 2004.
  21. Wilke CM, Hall BK, Hoge A, Paradee W, Smith DI, Glover TW. FRA3B extends over a broad region and contains a spontaneous HPV16 integration site: Direct evidence for the coincidence of viral integration sites and fragile sites. Hum Mol Genet 1996;5:187–95.[Abstract/Free Full Text]
  22. Becker NA, Thorland EC, Denison SR, Phillips LA, Smith DI. Evidence that instability within the FRA3B region extends four megabases. Oncogene 2002;21:8713–22.[CrossRef][Medline]
  23. Thorland EC, Myers SL, Gostout BS, Smith DI. Common fragile sites are preferential targets for HPV 16 integration in cervical tumors. Oncogene 2003;22:1225–37.[CrossRef][Medline]
  24. Corbin S, Neilly ME, Espinosa R III, Davis EM, McKeithan TW, Le Beau MM. Identification of unstable sequences within the common fragile site at 3p14.2: implications for the mechanism of deletions within fragile histidine triad gene/common fragile site at 3P14.2 in tumors. Cancer Res 2002;62:3477–84.[Abstract/Free Full Text]
  25. Greenspan DL, Connolly DC, Wu R, et al. Loss of FHIT expression in cervical carcinoma cell lines and primary tumors. Cancer Res 1997;57:4692–8.[Abstract/Free Full Text]
  26. Muller CY, O'Boyle JD, Fong KM, et al. Abnormalities of fragile histidine triad genomic and complementary DNAs in cervical cancer: Association with human papillomavirus type. J Natl Cancer Inst 1998;90:433–9.[Abstract/Free Full Text]
  27. Nakagawa S, Yoshikawa H, Kimura M, et al. A possible involvement of aberrant expression of the FHIT gene in the carcinogenesis of squamous cell carcinoma of the uterine cervix. Br J Cancer 1999;79:589–94.[CrossRef][Medline]
  28. Siprashvili Z, Sozzi G, Barnes LD, et al. Replacement of Fhit in cancer cells suppresses tumorigenicity. Proc Natl Acad Sci U S A 1997;94:13771–6.[Abstract/Free Full Text]
  29. Ji L, Fang B, Yen N, Fong K, Minna JD, Roth JA. Induction of apoptosis and inhibition of tumorigenicity and tumor growth by adenovirus vector-mediated fragile histidine triad (FHIT) gene overexpression. Cancer Res 1999;59:3333–9.[Abstract/Free Full Text]
  30. Sard L, Accornero P, Tornielli S, et al. The tumor-suppressor gene FHIT is involved in the regulation of apoptosis and in cell cycle control. Proc Natl Acad Sci U S A 1999;96:8489–92.[Abstract/Free Full Text]
  31. Roz L, Andriani F, Ferreira CG, Giaccone G, Sozzi G. The apoptotic pathway triggered by the FHIT protein in lung cancer cell lines is not affected by Bcl-2 or Bcl-x(L) overexpression. Oncogene 2004;23:9102–10.[CrossRef][Medline]
  32. Fong LYY, Fidanza V, Zanesi N, et al. Muir-Torre-like syndrome in FHIT deficient mice. Proc Natl Acad Sci U S A 2000;97:4742–7.[Abstract/Free Full Text]
  33. Dumon KR, Ishii H, Fong LYY, et al. FHIT gene therapy prevents tumor development in FHIT deficient mice. Proc Natl Acad Sci U S A 2001;98:3346–51.[Abstract/Free Full Text]
  34. Fujishita T, Doi Y, Sonoshita M, et al. Development of spontaneous tumours and intestinal lesions in FHIT gene knockout mice. Br J Cancer 2004;91:1571–4.[Medline]
  35. Huebner K, Druck T, Siprashvili Z, Croce CM, Kovatich A, McCue PA. The role of deletions at the FRA3B/FHIT locus in carcinogenesis. Recent Results Cancer Res 1998;154:200–15.[Medline]
  36. Sozzi G, Pastorino U, Moiraghi L, et al. Loss of FHIT function in lung cancer and preinvasive bronchial lesions. Cancer Res 1998;58:5032–7.[Abstract/Free Full Text]
  37. Zochbauer-Muller S, Wistuba II, Minna JD, Gazdar AF. Fragile histidine triad (FHIT) gene abnormalities in lung cancer. Clin Lung Cancer 2000;2:141–5.[Medline]
  38. Zochbauer-Muller S, Fong KM, Maitra A, et al. 5'CpG island methylation of the FHIT gene is correlated with loss of gene expression in lung and breast cancer. Cancer Res 2001;61:3581–5.[Abstract/Free Full Text]
  39. Virmani AK, Muller C, Rathi A, Zochbauer-Muller S, Mathis M, Gazdar AF. Aberrant methylation during cervical carcinogenesis. Clin Cancer Res 2001;7:584–9.[Abstract/Free Full Text]
  40. Le Beau MM, Drabkin H, Glover TW, et al. An FHIT tumor suppressor gene? Genes Chromosomes Cancer 1998;21:281–9.[CrossRef][Medline]
  41. Croce CM, Sozzi G, Huebner K. Role of FHIT in human cancer. J Clin Oncol 1999;17:1618–24.[Abstract/Free Full Text]
  42. Aveyard JS, Knowles MA. Measurement of relative copy number of CDKN2A/ARF and CDKN2B in bladder cancer by real-time quantitative PCR and multiplex ligation-dependent probe amplification. J Mol Diagn 2004;6:356–65.[Abstract/Free Full Text]
  43. Mihaila D, Gutierrez JA, Rosenblum ML, Newsham IF, Bögler O, Rempel SA. Meningiomas: analysis of loss of heterozygosity on chromosome 10 in tumor progression and the delineation of four regions of chromosomal deletion in common with other cancers. Clin Cancer Res 2003;9:4435–42.[Abstract/Free Full Text]
  44. Guo Z, Wu F, Asplund A, et al. Analysis of intratumoral heterogeneity of chromosome 3p deletions and genetic evidence of polyclonal origin of cervical squamous carcinoma. Mod Pathol 2001;14:54–61.[CrossRef][Medline]
  45. Stein CK, Glover TW, Palmer JL, Glisson BS. Direct correlation between FRA3B expression and cigarette smoking. Genes Chromosomes Cancer 2002;34:333–40.[CrossRef][Medline]
  46. Ferber MJ, Thorland EC, Brink AA, et al. Preferential integration of human papillomavirus type 18 near the c-myc locus in cervical carcinoma. Oncogene 2003;22:7233–42.[CrossRef][Medline]
  47. Mimori K, Druck T, Inoue H, et al. Cancer-specific chromosome alterations in the constitutive fragile region FRA3B. Proc Natl Acad Sci U S A 1999;96:7456–61.[Abstract/Free Full Text]
  48. Hopman AHN, Smedts F, Dignef W, et al. Transition of high-grade intraepithelial neoplasia to microinvasive carcinoma is characterized by integration of HPV-16/18 and numerical chromosome abnormalities. J Pathol 2004;202:23–33.[CrossRef][Medline]
  49. Rassool FV, Le Beau MM, Neilly ME, van Melle E, Espinoza R 3rd, McKeithan TW. Increased genetic instability of the common fragile site at 3p14 after integration of exogenous DNA. Am J Hum Genet 1992;50:1243–51.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
G. E. Carpagnano, M. P. Foschino-Barbaro, A. Spanevello, O. Resta, F. Carpagnano, G. Mule, R. Pinto, S. Tommasi, and A. Paradiso
3p Microsatellite Signature in Exhaled Breath Condensate and Tumor Tissue of Patients with Lung Cancer
Am. J. Respir. Crit. Care Med., February 1, 2008; 177(3): 337 - 341.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
O. Kujan, R. Oliver, L. Roz, G. Sozzi, N. Ribeiro, R. Woodwards, N. Thakker, and P. Sloan
Fragile Histidine Triad Expression in Oral Squamous Cell Carcinoma and Precursor Lesions.
Clin. Cancer Res., November 15, 2006; 12(22): 6723 - 6729.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Holschneider, C. H.
Right arrow Articles by Karlan, B. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Holschneider, C. H.
Right arrow Articles by Karlan, B. Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation