Bleomycin sensitivity in patients with familial and sporadic polyposis: a pilot study

Bleomycin sensitivity in patients with familial and sporadic polyposis: a pilot study

Magaly M. Sales¹, Edmundo J. de Lucca¹, Seizo Yamashita² and Luis Henrique Cury Saad³

¹Departamento de Genética, Instituto de Biociências, UNESP, 18618-000 Botucatu, SP, Brasil.

²Departamento de Doenças Tropicais e Diagnóstico por Imagem and ³Departamento de Cirurgia e Ortopedia, Faculdade de Medicina, UNESP, 18618-000 Botucatu, SP, Brasil. Send correspondence to E.J.L.

ABSTRACT

Human peripheral blood lymphocytes from 10 patients with familial adenomatous polyposis (FAP) showed a significantly higher incidence of chromatid breaks when compared to cells from 10 normal individuals, after exposure to bleomycin (BLM) during the G2 phase. However, no significant increase in bleomycin sensitivity was observed in lymphocytes from 10 patients with sporadic adenomatous polyps (AP) vs. 10 normal individuals (P = 0.67). Individuals that exhibited an average number of chromatid breaks per cell higher than 0.80 were considered sensitive to the drug. No control showed susceptibility to BLM, as compared to 3 out of 20 patients.

INTRODUCTION

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome, with more than 90% penetrance. Affected people normally have over 100 adenomatous polyps (AP) in the colon and rectum, one or more of which inevitably progress to malignancy if untreated (Church et al., 1996). Recently, the gene for FAP was cloned, sequenced and localized in the long arm of chromosome 5q21-23 (Bodmer et al., 1987).

A number of investigators have reported that fibroblasts and lymphocytes from patients with colon and rectum adenomatosis have an increased level of chromatid aberrations (gaps, chromatid breaks, and chromatid exchanges) and chromosome aberrations (polyploidy, aneuploidy, dicentrics, and rings) to mutagenic agents (Hori et al., 1980; Miyaki et al., 1982; Heim et al., 1985; Parshad et al., 1985; Delhanty and Cooke; 1989). Thus, it appears that defective DNA repair in FAP cells may be responsible for susceptibility to colorectal cancer.

Bleomycin (BLM) is a radiomimetic chemical that has been used to induce DNA breaks in lymphocyte blood cultures in order to test for DNA repair capacity (Hsu et al., 1989). Hsu et al. (1989) related that among 313 cancerous individuals (colon, head/neck and lung cancer) exposed to BLM, approximately 70% showed an average number of chromatid breaks per cell higher than 0.80, while in 335 normal individuals from the control group this proportion was 22%. The authors suggested that mutagen sensitivity plays an important role in carcinogenesis of organs and tissues that have direct contact with the external environment (respiratory, digestive, and integumentary systems).

MATERIAL AND METHODS

Peripheral blood samples from patients with benign large bowel tumors were provided by the Department of Tropical Diseases and Imaging Diagnosis, School of Medicine, UNESP, Botucatu, SP, Brazil. Only individuals who had a complete colonoscopy were considered for this study. Ten subjects with familial adenomatous polyposis (four females and six males), 10 with sporadic adenomatous polyps (five females and five males), and 20 normal controls, matched by age and sex, were cytogenetically studied. Age distribution of patients ranged from 25 to 80 and in the control group, between 25 and 82 years.

Two simultaneous short-term cultures (72 h) for each individual were set up in RPMI 1640 media supplemented with 20% fetal bovine serum and phytohemagglutinin (PHA). Five hours prior to harvest, BLM (Blenoxane, Bristol Laboratories) was added in a final concentration of 0.03 U/ml to one of the cultures. One hour prior to harvest, 0.05 ml of colchicine (0.0016%) was added. The harvest procedure and slide preparation method followed conventional cytogenetic protocol. One hundred cells were examined and the number of chromatid breaks recorded from each culture (with and without the drug). The mean number of breaks per cell (br/c) was used to estimate sensitivity to the mutagen.

We followed the criteria proposed by Hsu (1987) and scored only chromatid breaks. Therefore, chromatid gaps or attenuated regions were disregarded, as well as cells with more than 12 br/c. According to Hsu (1987), counting chromatid gaps increases the total frequency of lesions scored, but information on sensitivity to bleomycin is not substantially altered.

Under our experimental conditions, BLM induced a large number of chromatid breaks but very few chromatid exchanges (Figure 1). Each chromatid break was considered as one break and each chromatid exchange, as two breaks. Chromosome aberrations (rings, dicentric, and acentric fragments) were considered spontaneous aberrations that occurred during the past cell generations or as late as the G1 phase of the present cell cycle. These aberrations were recorded but not added to the frequencies of chromatid breaks.

Figure 1
- A and C: Chromatid breaks (small arrows); A, B and D: complex chromatid exchanges (large arrows).

Individuals who showed an average number of br/c higher than 0.80 were considered sensitive to the drug (Hsu et al., 1985), and subjects that presented values of br/c greater than 1.00 were considered hypersensitive (Hsu et al., 1989).

Statistical analysis

The median test with Fisher's exact test (Siegel, 1975) was employed to compare mean values of breaks per cell between patients with FAP and AP vs. control groups, in the cultures with and without BLM induction. The criterion for significance was P < 0.05. The homogeneous test (Gattás, 1978) was used to compare bleomycin-induced break distribution in patient and control chromosome groups. The Goodman test (Goodman, 1964) was used to compare bleomycin-induced break proportion in male and female patient (FAP + AP) and control in chromosome groups, at the 0.05 level of significance.

RESULTS

No control individual showed chromatid exchanges. The majority of aberrations recorded were chromatid breaks. The frequencies of spontaneous breaks per cell varied from zero to 0.10 in patients with polyps and from zero to 0.04 in normal controls (Table I). Only one individual showed a high spontaneous break frequency. Among patients with FAP, two individuals showed sensitivity to BLM. Among patients with adenomatous polyps (tubular adenoma, villous adenoma and tubulovillous adenoma), only one was sensitive to the drug.

Table I
- Pathological diagnosis, sex, age (years) and mean breaks/cell frequency in lymphocytes with and without bleomycin (100 cells analyzed per treatment) of patients with polyps and normal control.

Patient	Pathological diagnosis	Sex	Age (year)	Breaks per cell		Control	Sex	Age (year)	Breaks per cell
				-/BLM	+/BLM				-/BLM	+/BLM
1	FAP	F	33	0.10	0.87	1	F	33	0.00	0.48
2	FAP	F	45	0.04	0.53	2	F	47	0.00	0.20
3	FAP	F	49	0.05	0.68	3	F	49	0.00	0.44
4	FAP	F	61	0.00	0.51	4	F	62	0.00	0.44
5	FAP	M	25	0.02	0.30	5	M	25	0.02	0.26
6	FAP	M	35	0.00	0.50	6	M	37	0.04	0.41
7	FAP	M	39	0.02	0.73	7	M	39	0.03	0.38
8	FAP	M	40	0.00	0.22	8	M	42	0.01	0.23
9	FAP	M	42	0.04	0.52	9	M	44	0.00	0.19
10	FAP	M	47	0.03	1.02	10	M	49	0.00	0.24
11	TVA	F	52	0.01	0.53	11	F	53	0.01	0.32
12	VA	F	55	0.00	0.26	12	F	58	0.00	0.25
13	VA	F	58	0.02	0.34	13	F	58	0.03	0.40
14	TVA	F	66	0.01	0.47	14	F	68	0.00	0.25
15	TA	F	80	0.01	0.53	15	F	82	0.02	0.46
16	TVA	M	61	0.02	0.92	16	M	61	0.03	0.76
17	HP	M	62	0.00	0.31	17	M	61	0.01	0.35
18	TA	M	71	0.01	0.37	18	M	71	0.02	0.62
19	TVA	M	72	0.02	0.27	19	M	71	0.01	0.46
20	TVA	M	77	0.02	0.50	20	M	76	0.00	0.31

FAP, Familial adenomatous polyposis; Adenomas: Tubular adenoma (TA), Villous adenoma (VA) and Tubulovillous adenoma (TVA); HP; Hyperplastic polyps; -/BLM: without bleomycin; +/BLM with bleomycin.

No significant difference in frequency of chromatid breaks was observed between patients with familial polyposis (P = 0.19) and adenomatous polyps (P = 0.67) vs. control groups in cultures without BLM (median test). However, a significant difference was observed between the group of patients with FAP and normal controls after treatment with bleomycin (P = 0.0027). The other AP patients were not significantly different from the control after exposure to BLM (P = 0.67).

The distribution of bleomycin-induced breaks in chromosome groups 1, 2, 3, B, C, D, E, F and G was similar in male patients with polyps (FAP + AP) and normal controls (c² = 9.9869, P = 0.27), but it was discordant in females (c² = 18.0104, P = 0.02) (data not shown). The proportion of bleomycin-induced breaks in chromosome groups 1, 3, B and C of female patients (FAP + AP) was significantly higher when compared with the proportion in female controls (Goodman test).

No significant difference was observed in the distribution of bleomycin-induced breaks in chromosome groups of patients with familial polyposis (c² = 8.9487, P = 0.35) and adenomatous polyps (c² = 9.1060, P = 0.33) vs. normal controls.

DISCUSSION

Knudson (1985) has suggested that a genetically determined elevated carcinogen sensitivity might be the underlying cause of many cancers that are now presumed to be induced by environmental factors. One way to detect an otherwise hidden constitutional chromosomal instability might be to stress cells in vitro with a mutagen (Hsu, 1983).

Several reports have indicated a constitutional instability of the genome in patients with FAP and Gardner's syndrome (GS = FAP with additional extracolonic lesions) under in vitro conditions (Delhanty et al., 1983; Gardner et al., 1982). In addition, chromosome instability has been evaluated in lymphocytes and fibroblasts of FAP and GS patients after mutagen induction.

The purpose of our investigation was to evaluate the effect of bleomycin on late S-G2 chromosomes in patients with FAP and also patients with sporadic adenomatous polyps. FAP patients showed a highly significant incidence of chromatid breaks when compared with cells from normal individuals, after exposure to BLM (P = 0.0027); in contrast, the levels of chromatid breaks in the AP lymphocytes were not different from those of control lymphocytes, after exposure to BLM (P = 0.67). Our results are in agreement with those of Heim et al. (1985), Hori et al. (1980), and Delhanty and Cooke (1989), who observed a significantly increased level of chromatid-type damage in FAP lymphocytes and/or fibroblasts exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). However, no evidence for increased sensitivity to mitomycin C (MMC) was found in lymphocytes and fibroblasts from patients with FAP (Friedman et al., 1982; Heim, 1985; Mazzullo et al., 1988).

In addition to its role in checkpoint control, the p53 tumor suppressor gene is implicated in activating an alternative pathway leading to programmed cell death (apoptosis). This pathway can be activated by treatment with physical or chemical agents. Hence, cell death is an important mechanism to minimize the mutagenic consequences that result from the survival of heavily damaged cells (Lane, 1994; Weinstein et al., 1995). In the study presented here, there was a statistically significant difference between FAP patients and controls, after exposure to BLM. It is possible that lymphocytes from FAP patients with mutant forms of p53 exhibited a genomic instability to the effects of bleomycin, presumably because they escaped apoptosis and proliferated despite DNA damage.

The present results, together with findings by others, suggest that bleomycin sensitivity by itself probably does not play a significant role as a pathway to carcinogenesis since very few cancer patients are exposed to this drug. It may, however, serve as a marker or an indicator of a cell defect responsible for the disease or coadjuvant to its development.

ACKNOWLEDGMENTS

We would like to thank the Department of Tropical Diseases and Imaging Diagnosis, Unesp School of Medicine, for providing the patient blood samples, and Adalberto José Crocci, Ph.D, of the Department of Statistics, Bioscience Institute, Botucatu, SP, Brazil. This work was supported by grants from FAPESP (No. 92/3143-9) and CNPq. Publication supported by FAPESP.

RESUMO

Inúmeros estudos têm mostrado que fibroblastos de pacientes com adenomatose hereditária de cólon e reto, que inclui polipose adenomatosa familial (FAP) e a síndrome de Gardner, apresentam uma freqüência aumentada de aberrações cromossômicas após exposição a agentes físicos ou químicos, quando comparados aos controles normais. Para determinar a sensibilidade de linfócitos de pacientes com FAP e também com pólipos adenomatosos esporádicos (AP) usou-se o radiomimético bleomicina (BLM). Foram estudados citogeneticamente 10 indivíduos com AP, 10 com FAP e 20 controles normais, pareados por sexo e idade. Indivíduos que apresentaram valores médios de quebras cromatídicas por célula superiores a 0,80 foram considerados sensíveis à droga. Observou-se uma diferença significativa entre pacientes com FAP e controles quanto às freqüências de quebras cromatídicas nos linfócitos tratados na fase G2. Entretanto, nenhuma diferença significativa foi observada entre pacientes com AP e controles quanto às freqüências de quebras cromatídicas nos linfócitos tratados. Nenhum indivíduo do grupo controle foi sensível à BLM e, entre os 20 pacientes, três mostraram suscetibilidade à droga. Não foi encontrada diferença significativa quanto a resposta à bleomicina entre indivíduos do sexo masculino e feminino. Entretanto, a distribuição de quebras induzidas por bleomicina em cada grupo cromossômico não foi similar nos pacientes do sexo feminino e controles normais. É possível que a sensibilidade cromossômica à BLM encontrada nos pacientes com FAP esteja relacionada a deficiência de reparo de DNA.

(Received July 25, 1997)

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Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999

History

• Received
  25 July 1997