Nurhan Cucer

Condensed chromatin surface and NORs surface enhancement in mitogen-stimulated lymphocytes of Down syndrome patients

Mitogen-stimulated lymphocytes of 20 Down syndrome (DS) patients with regular trisomy 21 contain more condensed chromatin surface (11.28 +/- 2.64 % of the total nuclear surface: mean +/- SD) and more nucleolus organiser regions surface (13.21 +/- 3.45 %) than that of 12 healthy controls: (8.84 +/- 2.23 and 9.12 +/- 2.33 %, reciprocally). The source of this peculiarity has been investigated. A computer program was designed for the planimetric measurement of the condensed chromatin surface (CCs)/ total nuclear surface(TNs) and the nucleolus organiser regions surface (NORss) /TNs proportions in interphase nuclei. CCs/TNs and NORss/TNs of 100 maximally activated nuclei (MANs) were measured for each patient and control case. The difference was found highly significant (P<0.01). Nuclei with a diameter of >/= 17 micrometer measured on the slide (in flattened state) were considered as maximally activated nuclei (MANs). NORss/TNs enhancement and fluorescent in situ hybridisation (FISH) studies in MANs of DS patients indicate that this phenomenon is due to the over-expression (or lack of downregulative mechanism) of NORs (rDNA) to some extent, including the NOR of the supernumerary chromosome 21. No statistical difference was observed between 12 healthy controls and 5 Robertsonian translocation type of DS Patients (where the two involved NORs are missing) when the two parameters were considered.

Sister-chromatid exchange inducing effect of smokeless tobacco using on T-lymphocyte chromosomes

A kind of a smokeless tobacco (Maras powder) is widely used instead of cigarettes in the South Eastern region of Turkey. In this study we investigated the sister-chromatid exchange (SCE) inducing effect of this powder on the chromosomes of its users compared with smokers and nonsmokers using standard cell culture methods and SCE staining techniques. Average SCE per metaphase and total SCEs increased significantly among both smokeless tobacco users and smokers compared to nonsmokers (p < 0.01). However, the effect is significantly lower in smokeless tobacco users than in smokers (p < 0.05).

The Evaluation of Argyrophilic Nucleolar Organizing Region Proteins in Fine-Needle Aspiration Samples of Thyroid

Argyrophilic nucleolar organizing region associated proteins (AgNORs) have been shown to be of interest in a variety of different diseases including thyroid disorders. Our aim was to distinguish benign thyroid lesions from papillary thyroid carcinoma (PTC) via AgNOR count and with a new approach, via AgNOR surface area/total nuclear surface area (NORa/TNa) proportions in the nuclei on fine-needle aspiration (FNA) materials. Thirty patients (eight men and 22 women) whose FNA was compatible with benign lesion and 26 patients (eight men and 18 women) whose FNA was compatible with PTC were included in the study. Fine-needle aspiration materials were stained for AgNOR detection according to a specific protocol. One hundred nuclei per individual have been evaluated, and AgNOR number and NORa/TNa proportions of individual cells were measured and calculated by using a computer program. Patients with PTC had significantly (p < 0.001) higher AgNOR count (4.6 ± 1.2%) than in the patients with benign lesions (2.0 ± 0.5%). Additionally, patients with PTC had significantly (p < 0.001) higher NORa/TNa (13.4 ± 2.4) than in the patients with benign lesion (5.7 ± 1.0). Modified method of AgNOR staining is an easy and reliable method for evaluating proliferation activity of cells in malignant and benign thyroid lesions and it may contribute to routine cytopathology in inconclusive situations.

Methylation status of CpG islands at sites -59 to +96 in exon 1 of the BRCA2 gene varies in mammary tissue among women with sporadic breast cancer.

Germline mutations of the BRCA2 gene on chromosome 13q12-13 predispose humans to the development of earlyonset breast cancer and ovary cancer (Wooster et al. 1995). Most germline mutations in BRCA2 are predicted to result in truncation (Wooster et al. 1995; Tavtigian et al. 1996) and hence, inactivates the critical functions of the encoded protein (Shamoo 2003). Tumours arising in carriers of BRCA2 germline mutations usually exhibit loss of heterozygosity (LOH) of chromosome 13q polymorphic markers flanking BRCA2. The allele lost is the wild-type allele inherited from the nonmutation-carrying parent (Collins et al. 1995), a pattern that is characteristic of a tumour-suppressor gene and that is predicted to result in the absence of the functional protein in the tumour cell. In addition to germline mutations, many cancer predisposition genes including RB1, p53, NF-2, APC, WT1 (Fearon 1997), VHL (Shuin et al. 1994) NF-1 (Li et al. 1992) and MTS1 (Caldas et al. 1995) are somatically mutated in sporadic cancers. These somatic mutations are usually associated with a high frequency of LOH near the susceptibility gene, relevant to the sporadic cancers. Loss of heterozygosity at the BRCA2 locus has been observed in 30%–40% of sporadic primary breast cancers (Callens et al. 2003) and approximately 50% of sporadic primary ovarian cancers (Takahashi et al. 1996). However, exhaustive analyses of many sporadic breast, ovarian and other cancers have indicated that somatic mutations in BRCA2 gene are very rare (Collins et al. 1997).

Argyrophilic nucleolar organizing region associated protein synthesis in hair root cells of humans at different developmental stages and sex.

Abstract Argyrophilic nucleolar organizing region (AgNORs) associated proteins are important for cell proliferation and various diseases. We investigated AgNOR protein synthesis in hair root cells of males and females at different ages using two-dimensional image analysis. Experiments were performed on 58 healthy male and 24 healthy female volunteers in three groups according to age and sex. Hair root cells obtained from hair follicles were stained with silver. Total AgNOR number/total nuclear number (TAN/TNN) and total AgNOR area/nuclear area (TAA/NA) for each nucleus were analyzed. The only significant difference was observed in TAA/NA values for males and females from 6 to 12 years old. We suggest that the difference is due to high NOR activity caused by increased growth hormone production in hair root cells.

Detection and comparison of cut‐off values for total AgNOR area/nuclear area and AgNOR number/nucleus in benign thyroid nodules and normal thyroid tissue

Argyrophilic nucleolar organising region associated proteins (AgNORs) are of interest in a variety of diseases including thyroid disorders. We have investigated the cut‐off values for AgNOR count and with a new approach, Total AgNOR area/nuclear area (TNORa/Na) proportions to discriminate thyrocytes obtained from benign thyroid nodules and normal thyroid tissue. Thirty patients whose fine needle aspiration (FNA) materials were compatible with a benign lesion (named as goiter group) and 30 controls (subjects with normal thyroid tissue) were included. In the control group, biopsy material was obtained from histologically normal thyroid gland operated on because FNA material was compatible with papillary thyroid carcinoma (PTC). These samples were stained for AgNOR and 100 nuclei per individual were examined. Both AgNOR values for individual cells and cut‐off values were detected for each group. Patients with goiter had significantly (P < 0.001) higher AgNOR count (2.1 ± 0.6%) and TNORa/Na (6.1 ± 1.5%) than the control groups (1.4 ± 0.2 and 3.0 ± 0.4, respectively). The cut‐off values were <1.5 for AgNOR count and <3.8 for TNORa/Na in thyrocytes of the controls. This modified method is an easy and reliable method for discriminating various thyroid disorders, including the differentiation of benign thyroid nodules from malignant ones. It also helps to discriminate thyrocytes obtained from benign nodules from normal thyroid tissue, aiding accurate localisation of sampling in FNA material.

Condensed chromatin enhancement in the phytohemagglutinin-stimulated lymphocyte interphase nuclei of two xeroderma pigmentosum patients and artificial chromatin condensation of healthy cell nuclei by UV irradiation.

To the Editor: Condensed chromatin, in 0.5% v/v phytohemagglutinin (PHA, Difco Catalog no. 0528-56-6) stimulated lymphocytes from two xeroderma pigmentosum ( XP) patients (sibs born to first-degree consanguineous parents, 8 years/female and 7 years/male), after 72 h of culture occupies more than 12% of the cells’ nucleus surface, whereas this proportion is only -5% for 12 healthy subjects and -6.5% for 900 routinely analyzed patients in our cytogenetics laboratory under the same culture conditions. The percentage of cells containing an increased level of the condensed chromatin (equal to or higher than 20% of the nuclear surface) in two XP patient was 69 and 8 1, respectively. However, on random choice and measurement, the respective condensed chromatin proportions were nearly 15% and 17%. PHA concentration should be constant during these experiments, because we observed a positive parabolic relationship between PHA concentration in the culture media and the condensed chromatinized surfaces in the nuclei. There was also no constant/consistent condensed chromatin level in cells from different healthy subjects when these cells were cultured in the same medium. Two methods were used to measure condensed chromatin in the nucleus:

Comparison of fine needle aspiration biopsy and paraffin embedded tissue sections for measuring AgNOR proteins

Abstract Paraffin embedded tissue sections and fine needle aspiration biopsy (FNAB) are important methods for diagnosis. We compared thyroid tissue obtained by FNAB to paraffin embedded sections to determine whether there were differences in detection of the amounts of argyrophilic nucleolar organizing region (AgNOR) proteins. Twenty-two patients with papillary thyroid carcinoma were included in the study. Slides were prepared with both FNAB tissue and 3 μm sections of paraffin embedded tissue, and stained for AgNOR. One hundred nuclei per individual were evaluated; total AgNOR number/nucleus (TAn/TNn) and total AgNOR area/nuclear area (TAa/TNa) of individual cells were determined. Mean TAn/TNn and TAa/TNa values were 4.800 ± 1.118 and 13.382 ± 2.612, respectively, for FNAB samples; corresponding values were 2.406 ± 0.649 and 8.49 ± 0.893, respectively, for paraffin embedded sections. The differences between FNAB materials and paraffin embedded tissue sections were significant for the mean TAn/TNn and TAa/TNa values. Significant differences in the amounts of AgNOR protein detected were found between FNAB and paraffin embedded tissue sections.

Argyrophilic nucleolar organizing region associated protein synthesis for cytologic discrimination of follicular thyroid lesions

Abstract Fine needle aspiration biopsy (FNAB) of the thyroid gland is an important tool for preoperative diagnosis; however, its benefit is limited for follicular lesions. Nucleolar organizer regions (NORs) are ribosomal gene regions that stain with silver (Ag) when they are active. These regions can be used to differentiate neoplastic and non-neoplastic lesions. We used a new AgNOR technique to investigate FNAB of cases diagnosed as follicular adenoma and carcinoma. Fourteen cases of follicular thyroid carcinoma (FTC) and 28 cases of thyroid follicular adenomas (FA) were stained using the silver NOR-associated protein (AgNOR) technique. One hundred nuclei per sample were examined, AgNORs were counted, and the total AgNOR area/nuclear area (TNORa/Na) ratio of each cell was calculated. We found that cases with FTC had significantly higher TNORa/Na than cases of FA. Also, cases with FTC had significantly higher AgNOR counts than cases with FA. AgNOR counting may help discriminate FTC and FA by routine cytopathology before surgery.

Staining human lymphocytes and onion root cell nuclei with madder root.

We performed staining experiments on cells using natural dyes and different mordants using techniques that are used for wool and silk dyeing. The natural dye sources were madder root, daisy, corn cockle and yellow weed. Ferrous sulfate, copper sulfate, potassium tartrate, urea, potassium aluminum sulfate and potassium dichromate were used as mordants. Distilled water, distilled water plus ethanol, heptane, and distilled water plus methanol were used as solvents. All dye-mordant-solvent combinations were studied at pH 2.4, 3.2 and 4.2. The generic staining procedure was to boil 5–10 onion roots or stimulated human lymphocyte (SHL) preparations in a dye bath on a hot plate. Cells were examined at every half hour. For multicolor staining, madder-dyed lymphocytes were decolorized, then stained with Giemsa. The AgNOR technique was performed following the decolorization of Giemsa stained lymphocytes. Good results were obtained for both onion root cells and lymphocytes that were boiled for 3 h in a dye bath that included 4 g madder root, 4 g ferrous sulfate as mordant in 50 ml of 1:1 (v/v) methanol:distilled water. The pH was adjusted to 4.2 with 6 ml acetic acid. We conclude that madder root has potential as an alternative dye for staining biological materials.