Study of numerical aberrations of chromosome 1 by fluorescent in situ hybridization and DNA content by densitometric analysis on (pre)-malignant cervical lesions.

In an attempt to determine whether the fluorescent in situ hybridization (FISH) can be used as a rapid approach for the identification of aneuploidy in premalignant cervical smears, a centromeric probe for chromosome 1 was used. The results from the FISH experiments were compared with measurements of the overall DNA content obtained by means of an image analysis system. With progression to neoplasia, a decrease of the frequency of cells with two spots was observed, due to an increasing polysomy of chromosome 1. As far as the DNA content was concerned, an increasing DNA index and 5C-exceeding ratio (fraction of cells with a DNA content higher than 5C) was observed. Classification of the FISH results by a linear discriminant analysis revealed that 67.6% of the cases were classified in agreement with the CIN classification. These data suggest that chromosome 1 may be considered as a marker chromosome for pre-malignant cervical lesions and that the DNA content measurements are complementary to the FISH results.

Detection of premalignant stages in cervical smears with a biotinylated probe for chromosome 1.

Fluorescence in situ hybridization with (peri-)centromeric probes is an easy method to detect numerical aberrations in nonmitotic and mitotic cells. In this study, cervical smears of premalignant and malignant stages (26 controls, 15 CIN I, 12 CIN II, and 15 CIN III cervical smears) were analyzed for the presence of numerical aberrations of chromosome 1 with a centromeric DNA probe (1q12). With more severe stages a decrease of disomy was observed, merely due to a gain of extra copies of chromosome 1; in some cases, however, monosomy was detected. The frequencies of disomy for chromosome 1 ranged from 65.3% to 95.0% in the controls, from 71.3% to 94.3% in CIN I, from 59.2% to 91.5% in CIN II, and from 23% to 96.2% in CIN III. Polysomy ranged from 0% to 5.7% in the controls, from 0% to 14.4% in CIN I, from 0.9% to 30.8% in CIN II, and from 0.8% to 69.6% in CIN III. Monosomy ranged from 2.6% to 34.1% in the controls, from 0% to 17.5% in CIN I, from 3.6% to 27.5% in CIN II, and from 0.9% to 31.4% in CIN III. The results show that screening for aneuploidy of chromosome 1 allows a good discrimination between control samples and dysplasia. These data suggest that chromosome 1 may be a marker chromosome. They are in accordance with previous cytodensitometric analyses, where already in the preneoplastic stages an increased DNA content (polyploidization with subsequent aneuploidization) is observed.

Induction of micronuclei and karyotype aberrations during in vivo mouse skin carcinogenesis.

The induction of chromosome and/or genome mutations during the first steps of skin carcinogenesis was followed in male NMRI mice, treated with a 'two-stage' [9,10-dimethyl-1,2-benzanthracene (DMBA) + phorbol-12-myristate-13-acetate (TPA)], or a 'three-stage' [DMBA+methyl methanesulphonate (MMS) + phorbol-12-retinoate-13-acetate (RPA)] protocol. The scoring of micronuclei (MN) in basal and suprabasal keratinocytes allows a relatively fast in vivo estimation of clastogenic and aneugenic effects of various compounds and treatments. Relevant stages were then further analysed by karyotyping the in vivo treated keratinocytes that were allowed to divide during short in vitro cultivation. DMBA used as initiator in both protocols was able to induce MN. The well-known clastogen MMS had an acute but transient effect on MN induction when used alone or as convertor in the three-stage protocol. Neither the propagator RPA, nor the 'full-promotor' TPA, which can carry out conversion as well as propagation, induced statistically significant numbers of MN when applied on mouse skin. Combined treatments, DMBA+MMS and MMS+RPA, showed higher MN frequencies than when MMS treatments were given alone. The full carcinogenic protocols showed significant frequencies of MN but the time points of appearance differed, indicating that the accumulation of aberrations could be more important than the order of appearance. Karyotypic analysis of those stages where the MN assay detected genome and/or chromosome aberrations revealed no specific loss of chromosomes that might be directly related to the carcinogenic process. When chromosome loss and aberrations were both taken into consideration together, chromosomes 7 and 11 and surely 9, 17 and 18 were more frequently involved than others.

The influence of phenobarbital and butylated hydroxytoluene on the ploidy rate in rat hepatocarcinogenesis.

The effect of the 'promoters' phenobarbital (PB) and butylated hydroxytoluene (BHT) on the ploidy changes during hepatocarcinogenesis in rats was compared in a densitometric analysis of Feulgen-stained nuclei on paraffin-embedded tissue slices. The triphasic Gerlans protocol for liver-cancer induction was applied. Initiation with a single dose of diethylnitrosamine (DEN), and selection with 2-acetylamino-fluorene (2-AAF) combined with a proliferative stimulus (CCl4 administration), was followed by a treatment with PB or BHT for periods up to 22 weeks. Control animals received no treatment after the initiation and selection procedure. Despite intra- and inter-individual variations, an increase in the amount of 2N nuclei is found in the putative preneoplastic lesions of animals that received initiation and selection (I-S) and 3 weeks basal diet (BD). When the diet is supplemented with PB (after I-S), the increase of diploid nuclei starts earlier. At the time carcinoma arise (22 weeks PB treatment) a decrease in the frequency of 2N nuclei is found. BHT-treated animals which develop no carcinoma within the considered timespan, show a clear increased amount of 2N nuclei in the precancerous lesions only after 14 weeks treatment. It seems that there is a positive correlation between the outgrowth of putative preneoplastic foci and nodules in rat liver and an increase of diploid nuclei in these lesions. PB, as promoter used after initiation and selection, speeds up the development of carcinoma in rat liver, and therefore also the shift to diploidization in these rats starts earlier in comparison with I-S-treated rats. Although BHT does not promote liver carcinogenesis, an increase of diploid nuclei is also observed here during lesion formation. It may, therefore, be concluded that the phenomenon of diploidization is closely linked to and probably necessary for preneoplastic development, but that it is not an absolute indicator for neoplastic transformation.