Chromosome fragility in lymphocytes of women with cervical uterine lesions produced by human papillomavirus

Chromosome Fragility in Lymphocytes of Women with Cervical Uterine Lesions Produced by Human Papillomavirus C sar Paz-y-Mifio, Ligia Ocampo, Romeo Narvfiez, and Luis Narvfiez

We studied 30 w o m e n with cervical lesions that shawed human papillomavirus infection (IIPV). Cervical HPV infection was diagnosed by cytology, histology, i m m u n o h i s t o c h e m i s t r y , and electron microscopy, as well as by DNA viral hybridization in situ with 6, 11, 16, and 18 HPV types. Three groups of patients were studied: 15 women infected by HPV of 6 and 11 types with koilocytic lesions and benign ew~lution, 15 women infected by HPV of 16 and 18 types with koilocytic lesions and malignant evolution, and 15 normal women without cervical lesions who served as controls. For each group, c h r o m o s o m e fragility was studied in peripheral blood lymphocytes. Aphidicolin (AP) was used as a clastogenic agent at a concentration of 0.12 I~M. There were significant differences (P < 0.001) between the control population and the patients affected by HPV. There were also significant differences ( p < 0.001) between the two groups infected with HPV. Our findings support the concept that c h r o m o s o m e fragility could serve as a cytogenetic marker to measure evolution, prognosis, and treatment of cervical lesion associated with HPV. ABSTRACT:

INTRODUCTION

Considerable amounts of data about cancer-associated chromosome aberrations have been described in recent years [1-4]. A high incidence of numerical and structural chromosomal aberrations in peripheral blood lymphocytes in patients with cancer, leukemia, chromosome fragility syndromes, and viral infection has also been described [5-9]. Viral integration into the cell genome produces no specific cytogenetic manifestations, usually manifesting as altered mitotic indexes, polyploidy, gaps, breaks, and abnormal chromosomal figures [10-14]. Some types of human papillomavirus (HPV) are believed to be involved in development of 95% of preneoplasic and neoplasic lesions of the cervix [15-18]. The HPV group includes at least 50 different types that cause a number of abnormal growths, but the most commonly encountered types in lesions of the uterine cervix are 6, 11, 16, and 18. Fifteen percent of the cervical lesions on dysplasia of low degree that evolved favorably in patients is associ-

ated with types 6 and 11. Twenty-five percent of the cervical lesions with a malignant evolution is associated with types 16 and 18 [19-21]. Cervical HPV infections can be diagnosed by colposcopy, cytology, histology, immunohistochemistry, electron microscopy, and hybridization techniques [14]. Very little is known about chromosome damage produced by HPV as related to cervical lesions. The specific chromosomal sites of viral DNA integration have been identified by hybridization [12, 22, 23]. The HPV integration sites have also been observed to lie near or coincide with chromosome fragile sites, cancer chromosome breakpoints, and location of oncogenes [12, 24]. Evidence also indicates that c:hromosomes fragility in peripheral blood lymphocytes of an individual might be associated with virus infection [10-12, 14]. We report findings of aphidicolin-induced chromosome fragility in cultured peripheral blood lymphocytes from women with cervical lesions associated with HPV infection. MATERIALS AND METHODS

From Laboratorio de Gen~tica Humana, l)epartamento de Ciencias Biol6gicas, Facultad de Ciencias Exactos y Naturales, P. Universidad Catolica del Ecuador, Quita (C. P-y-M), Laboratorio de Citologta, Instituto Nacional del Cdncer, SOLCA, Quito-Ecuador (L. 0.; R. N.; L. N.). Address reprint requests to: Professor Dr. C~sar Paz-y-Mino, Laboratorio de Gen~tica Humana, Departamento de Ciencias Biol6gieas. Facultad de Ciencias, P. Universidad Cat61ica del Ecuador, P.O. Box 17-01-2184. Quito-Ecuador. Received May 28, 1991 ; accepted September 26, 199 I. © 1992 Elsevier Science Publishing Co., Inc.

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Forty-five women, ranging in age from 18 to 42 years and divided into three groups, were studied. The first group (G-II included 15 patients with cervical lesions (CL) affected by 6 and 11 HPV. The second group (G-III included 15 patients with CL caused by 16 and 18 HPV. The third, the control group (C-GI consisted of 15 healthy women. HPV infection was identified according to ~Vorld Health Organization [WHO) criteria (19871 [14]. Histologic preparations from each group of patients were analyzed by 173 Cancer Genet Cytogenet59:173-176 {1992} 0165-4608/92/$05.00

174

Table 1

C. Paz-y-Mifio et al.

Total of altered metaphases in all groups

Group I

1 II II Control Control Total

Medium type

No. of analyzed metaphases

N

1,5(}0

AP N AP N AP

1,500 1.500 1,500 1.500 1,500 9,000

RESULTS

Altered metal)hoses

%

6.q 297 90 500 28 32 1,016

4.6 19.8 6.0 33.3 1.9 2.1

Abbreriatiotas: N, RPMI normal medium: AP. RPMI medium with aphidicolin.

hybridization in situ. HPV types 6, 11. 16, and 18 were identified (DNA Probe-Based Essay, Enzie(:do Patho Gene DNA-HPV, Enzo Diagnostics). Five randomly HPV-posirive women u n d e r w e n t cervical biopsies. The (:ells were observed by electron microscopy [25] to confirm the presen(:e of HPV particles. Cytogenetic

Preparations

For cytogenetic preparations, patients who had some contact with physical or chemical agents that could induce chromosome fragility were not included. At the same time, healthy women of age similar to that of the patients were chosen to constitute the control group. Peripheral blood lympho(:ytes were seeded in two different media per individual: 1) A normal culture containing 5 ml of RPMI 1640 medium, 5% fetal calf serum, phytohemagglutinin, and penicillin-streptomycin in standard concentrations and 2) the above preparation with additi()n of aphidicolin (AP) in a final concentration of 0.12/.LM/ml. Cultures were maintained for 72 hours. Harvesting was performed according to standard techniques. Air-dried chromosome preparations were stained by the Giemsa method. One h u n d r e d metaphases per subject were scored. Statistical analysis was performed using the normal SD, and the significance of the observed differences between the test and control groups was calculated by Chisquare test.

Table 2

Group

Chromosomal aberrations such as breaks, gaps, acentric fragments, double minute (dmin) chromosmnes, rings, pulverizati()n and figures (classified ac(:ording to statements, of the International System for Human Cytogeneti(: Nomen(:lature 1985 [26]), were scored in 9,000 metaphase plates, from women with HPV infection and age-matched control women. Table 1 summarizes the cytogenetic findings of altered metaphases. The aberrations are expressed as absolute numbers of each type and also per 100 rectaphases. Table 2 includes all types of chromosomal alterati()ns noted in normal and AP (:ultures. Results from the statistical analysis are shown in Tables 3 and 4. Table 3 shows the mean. SD for the total metaphases analyzed for the normal culture, and the culture with AP for the three groups of patients: it also shows chi-square test (p-value) data. There was no difference between the results of the normal (:ulture and the AP culture for the control grou I) (p > 0.05). On the other hand. in the women with HPV infection, we noted differences between the normal culture and AP (:ulture (p < 0.01). Table 4 shows the comparisons between the groups. Evidently, patients with viral infection have more chromosome fragility than the control population; the statistical difference was significant (p < 0.001). There were significant statistical differences (p < 0.001) between the two groups of patients with HPV infection. This difference between groups I and II might be explained by the observations that patients affected with HPV 16 and 18 (group Ill who showed a high percentage (33.3%) of chromosomal alterations also have a higher probability to evolve unfavorably, according to some epidemiologic data.

DISCUSSION

We consider that our patients represent a good sampling group be(:ause the diagnosis of 6, 11, 16, and 18 HPV infection was confirmed by histologic, DNA in situ hybridization techniques and, in some patients, with electron microscopy. We used two different types of cell culture medium: a complete culture m e d i u m [normal medium) and another m e d i u m with AP. We used both (:ell cultures to ascertain the level of spontaneous chromosome fragility

Types of chromosome fragility in all analyzed groups Medium type

1 1

N AP

II II Control Control

N AP N AP

g

b

ct

cs

ct

cs

12

27

16

24

27 15 45 23 27

72 27 66 26 24

57 19 180 25 27

138 39 489 24 32

f

tr

qr

1

2

1

2

1

dmin

pVZ

1

3

3

Abbreviations: g, gaps: b, breaks; ct, chromatids; cs. chromosomes: I, fragments; tr, triradials: qr. quadriradials: dmin, double

minutes; pvz, pulverization;r, ring; other abbreviationsas in Table 1.

Chromosome Fragility and Papillomavirus

Table 3

175

Percentages of altered metaphases in each group

Group

Normal culture

Aphidicolin culture

Control I II

3.5 (±2.9 SD) 7.7 (+-3.9 SD) 9.7 (±3.5 SD)

8.8 (+-4.4 SD) 37.1 (+-12.4 SD) 31.2 (-+7.4 SD)

p-Value N/AP >0.05 <0.01 <0.01

more, the increased frequency of chromosome fragility in some patients with precancerous lesions supports this assumption: the increase of fragility may be important in the biologic behavior and progression of malignancy. Chromosome fragility may be a cytogenetic marker reflecting the evolution, prognosis, and treatment in women with HPV infection.

Abbreviationsas in Table 1.

and to induce fragility by AP. The latter is a diterpenoid mycotoxin and a eucaryotic DNA polymerase-a inhibitor, associated with DNA replication [27]. Use of this different m e d i u m has proven effective if we analyze the different fragility results in each group. Other investigators have also reported the validity of complete culture medium [28, 29]. The percentage of chromosomal spontaneous fragility in normal m e d i u m was not high and, to express it, we had to add a clastogen (AP). The chromosome fragility frequency in the peripheral blood lymphocytes was increased in patients with cervical lesions associated with HPV infection, in contrast to the control group. This fragility increase could be explained by the HPV infection, because viral genome integration into the host genome provides a background that predisposes to chromosome fragility. Another explanation may be that women infected with HPV have a chromosomal hypersensitivity to AP, like that observed in lymphocytes of subjects with cancer [1, 5, 6, 8, 9]. This fragility, according to other investigations [10, 12, 14, 22, 30, 31], is particularly evident in culture m e d i u m with a clastogen (AP). Even though the biologic meaning of chromosomal fragility in the development and progress of cancer remains unclear, a high chromosome aberration frequency may play a critical role in cancer predisposition or progression [2, 3, 5, 7, 32-34]. The increased frequency of chromosome fragility in the present investigation may indicate that chromosome fragility in women with HPV infection could result from genetic factors. Although HPV infections appear to interact synergistically with chemical or physical carcinogens in carcinoma development [18, 35, 36], individuals with increased chromosome fragility may be at a high risk to develop cancer [33, 37]. This appears to be true if we analyze the comparative data between groups I and II of women infected by HPV, which differences were highly significant (p < 0.001). Considering our results, the greater rate of chromosomal alterations (33.3%) observed in women with types 16 and 18 HPV infection, may reflect a higher risk of unfavorable evolution in cervical uterine lesions, as shown by epidemiologic data [19-21 ]. Further-

Table 4

Comparative analysis between different groups

Culture

C G/C G

C G/G I

C G/G II

G I/G II

N AP

p > 0.05 p > 0.05

p > 0.05 p < 0.001

p > 0.05 p < 0.001

p > 0.05 p < 0.001

A b b r e v i a t i o n s as in Table 1.

This work was partially supported by Science Area of "Sim6n Bolivar Fundation" of Quito. The authors thank the staff of the Human Genetics Laboratory of the Universidad Cat61ica del Ecuador: Augusta C6rdova, Sara Guti~rrez, Paola Leone, Ma. Serena Pefiaherrera, and Ma. Eugenia S~nchez; the authors also thank Professor Dr. Benjamin Ollgaard for correcting the manuscript.

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