Reconstructed three-dimensional images of flow-sorted nuclei hybridized with chromosome-specific probes.

We demonstrated that the three-dimensional (3-D) locational and morphological differences of chromosome 17 are dependent on each cell cycle phase in the clinical materials. Cell suspensions prepared from hypertrophied tonsil were hybridized with chromosome 17 whole painting probe or its centromeric probe and the probes were detected with fluorescein isothiocyanate. Then the cells were sorted from G(0+1), S-, and G(2+M)-phase fractions by flow cytometry and observed by confocal laser scanning microscopy to obtain the serial optical sections. The 3-D images were obtained by assembling these sections using a computerized image analysis device. The distribution of centromeric copies was analyzed statistically, and the data values were not a population of random distribution within a sphere. The copies were observed in the periphery of the nuclei in G(0+1)- and S-phase. The 3-D images revealed that chromosome 17 was oval in shape in the G(0+1)-phase nucleus, and was changing into a flame shape in the S-phase, with arms stretching out along the nuclear membrane, and looked bush shaped in G2-phase. The eccentric distribution of chromosome 17 in G(0+1)- and S-phase nuclei may reflect the optimal efficiency of incorporating and/or releasing essential materials and products.

Estimation of S-phase fraction in tumor tissue sections by immunohistochemical staining of PCNA.

We describe a method for measuring the size of the S-phase fraction in human tumor tissue sections using an antibody to PCNA (PC10). Although treatment with Triton X-100 before fixation extracted a large amount of PCNA from the cells even in frozen tissue sections, PCNA remained exclusively in S-phase cells. Immunohistochemical staining of PCNA after the detergent treatment allowed estimation of the S-phase fraction in solid tumors. The validity of the method was directly proven by double staining of bromodeoxyuridine (BrdU) and PCNA in HeLa cells. The PCNA-positive cells were identical with BrdUrd-positive cells after the detergent treatment. In contrast, almost all HeLa cells in the exponentially growing phase were positive for PC10 without treatment with Triton X-100.

Immunohistochemical detection of p21WAF1/CIP1 and p53 proteins in formalin-fixed paraffin-embedded tissue sections of squamous cell carcinoma of the skin.

Thirty-two cases of squamous cell carcinoma (SCC) of the skin were investigated as to the expression of p53 and p21 (WAF1/CIP1) using an immunohistochemical method. These cases were surgically resected or biopsied, tissue samples were then fixed in formalin and embedded in paraffin in the conventional way. Microwave heating was used for antigen retrieval. The primary monoclonal anti-p21 antibody and the monoclonal antibody against p53 were employed. The labeled-streptoavidin-biotin-peroxidase method was used for immunohistochemical staining. Of these, 30 cases showed overexpression of p53 staining, but normal epidermal cells were free of stain. p21 positive cells were detected faintly in the middle layer cells of normal epidermis. Of these, 30 cases showed overexpression of p21 staining. The staining pattern of p53 and p21 showed intratumoral heterogeneity in SCC. In general, there was the inverse relationship between p21 and p53 staining in tumors, namely p53 positive cells were p21 negative and vice versa. However, some of the tumor cells expressed both genes simultaneously. This study supports a hypothesis that p21 expression is regulated by p53, and that it is also regulated by an additional pathway(s) in SCC.

[Comparative genomic hybridization(CGH)].

A new molecular cytogenetic method, comparative genomic hybridization(CGH), was reviewed. CGH produces a map of DNA sequence copy number on a normal metaphase spread after hybridization with the mixture of tumor DNA and normal reference DNA, which are detected with different fluorochromes, respectively. Then the ratio of two fluorochromes are analysed with digital image analyzer and reveals a real copy number of the DNA sequences. CGH surveys entire chromosomes at a time and would clarify oncogenes and tumor suppressor genes, which had not known.

[Relationship between the cell cycle and production of hCG in choriocarcinoma cells].

Human choriocarcinoma cells in vitro (ENAMI-1) were exposed to various concentrations of MTX (10(-8)M, 10(-7)M, 10(-6)M, 10(-4)M) for 48 hr, and after removal of MTX, cells were harvested and measured by FCM every 24 hr for 96 hr. Although the growth of cells were inhibited in MTX more than 10(-7)M in concentration, hCG-beta levels were elevated markedly. It was showed that hCG positive cells were accumulated in G1-phase by flow cytometric two color analysis with monoclonal antibody. In the presence of MTX more than 10(-7)M in concentration, the cells were accumulated in S-phase. After removal of 10(-7)M MTX, S-phase cells migrated toward G2 + M phase on the other hand in the presence of MTX more than 10(-6) M in concentration cells stopped moving to G2 + M phase. Immunoperoxidase with monoclonal antibodies against BrdU and hCG-beta has got the same phenomena resulted by FCM. It was confirmed that hCG was an useful marker for choriocarcinoma.

[Recent problems on the endocrine and chemotherapy].

The endometrial carcinoma, as well as the breast cancers and some kinds of ovarian cancers, was considered to a hormone dependent carcinoma. Even if we tried to do the endocrine therapy using such as medroxy progesterone acetate (MPA), we could expect only 30% of anti-tumor effects on the endometrial carcinoma. Endocrine therapy was thought to have a different action mechanisms from the other anti-cancer drugs. Whereas cisplatin (CDDP) has strongly an effectiveness for ovarian cancers, but the drug resistance of cancer cells for CDDP was causing a serious problem. This paper will be discussed about the problems of the endocrine and chemotherapy from the point of view of cell cycle analysis. Additionally, we would like to describe about the new anticancer drug such as Taxol.

Applications of DNA flow cytometry and fluorescence in situ hybridization using a chromosome-specific DNA probe on paraffin-embedded tissue sections of primary malignant melanomas.

We have applied DNA flow cytometric analysis to paraffin-embedded tissue sections of primary malignant melanomas. Conventionally, flow cytometric analysis of paraffin-embedded tissue sections has been done by the method of Hedley et al. We added ultrasound treatment to the method of Hedley et al. and a lower value of coefficient of variation was shown. Furthermore, a new technique, fluorescence in situ hybridization with a chromosome-specific repetitive DNA probe, was used for the analysis of chromosomal numerical aberrations in the same paraffin-embedded tissue sections. The DNA flow cytometric analysis showed that in 8 cases six primary malignant melanomas were of the aneuploid pattern and two cases of lentigo maligna (melanoma in situ) were of the diploid pattern. By fluorescence in situ hybridization, the two cases with the diploid pattern had spots/nucleus of 1.28 and 1.12, and those with the aneuploid pattern had spots/nucleus from 2.01 to 2.27. Only one nodular melanoma in an aneuploid case showed spots/nucleus of 1.71. These data indicate that fluorescence in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors and may be useful for the study of tumor cell heterogeneity.

Immunohistochemical detection of Ki-67 in epithelial skin tumors in formalin-fixed paraffin-embedded tissue sections using a new monoclonal antibody (MIB-1).

The expression of the Ki-67 antigen was investigated in 44 epithelial skin tumors using an immunohistochemical technique on formalin-fixed, paraffin-embedded tissue sections. Microwave oven heating was employed for retrieval of the antigen in these tissue sections. The staining patterns varied among the epithelial skin tumors. The assessment of immunohistochemical staining was based upon the growth fraction (GF), defined as the number of Ki-67 positive cells divided by the total number of tumor cells counted and expressed as a percentage. GF was 9.7 +/- 3.1% in seborrheic keratosis, 19.5 +/- 2.9% in keratoacanthoma, 23.1 +/- 4.9% in basal cell carcinoma, 18.5 +/- 6.3% in actinic keratosis, 37.1 +/- 6.0% in Bowen's disease, and 32.9 +/- 10.5% in squamous cell carcinoma. There was a significant difference in GF between the keratoacanthoma and squamous cell carcinoma (p < 0.01). Actinic keratosis showed a relatively low GF, whereas Bowen's disease showed a high one. Furthermore, the GF tended to increase with tumor cell differentiation in squamous cell carcinoma: 23.7% (+/- 5.0) in well-differentiated, 35.0% (+/- 6.2) in moderately-differentiated, and 47.6% (+/- 4.5) in poorly-differentiated squamous cell carcinomas. Immunohistochemistry with MIB-1 may give useful additional information in the differential diagnosis of KA and SCC.

Cutaneous mixed tumor containing ossification, hair matrix, and sebaceous ductal differentiation.

A 58-year-old Japanese male presented with a cutaneous mixed tumor containing ossification and hair matrix differentiation on the left side of the chin. Histologically, the tumor consisted almost exclusively of apocrine-type epithelial ductal structures and chondroid stroma. Strands and aggregation of basaloid cells which contained keratinous cystic structures with a column of shadow cells arising from basophilic basaloid cells, sebaceous duct-like structures, and ossification in the stroma were also evident. These findings suggest that cutaneous mixed tumors with ossification and hair matrix differentiation are related to both the whole hair follicle and the sweat apparatus.

Neonatal lupus erythematosus occurring in identical twins.

The patients were one-month-old identical twins. Scaly erythema was noted mainly on the trunk, face, and scalp of one twin starting about three weeks after birth and starting about two weeks after birth in the other. The patients' courses were observed without treatment; the eruptions tended to disappear two months after birth. The mother had a past history of transient facial erythema. Both twins and mother were positive for antinuclear antibody, anti-SS-A antibody, and anti-SS-B antibody. The histopathological findings corresponded to those of discoid lupus erythematosus (DLE). The class of HLA typing revealed Cw3 in both twins, which is frequently observed in neonatal lupus erythematosus (NLE), and A24 in the mother, which is frequently observed in mothers of babies with NLE.

A case of systemic sclerosis associated with gastric cancer.

We report the case of a 47-year-old woman who began to experience stiffness and Raynaud's phenomenon of her fingers and toes as well as easy fatigability approximately one year before initial evaluation. Histopathologic examination revealed dense fibrosis and perivascular lymphoid cell infiltration in the dermis. The patient's diagnosis was systemic sclerosis (SSc). Esophageal sclerosis was not revealed, but an early type IIb carcinoma of the gastric antrum was noted by endoscopic examination. Other recent reports have discussed the association of SSc with various malignant carcinomas. We also reviewed the literature for associations between SSc and gastric cancer in Japanese patients.

Electron microscopic study of the colloid-like substance in solar elastosis.

We have made electron microscopic studies on the elastotic material of solar elastosis which developed in cutis rhomboidalis nuchae taken from 10 males, 50-83 years of age. It was revealed that this material of the cutis rhomboidalis nuchae contained a colloid-like substance consisting of both a fine granular and an amorphous component. The colloid-like substance closely resembled that which is seen in the adult-type colloid milium, and could not be distinguished from it under light microscopy. In view of the morphological similarity at the ultrastructural level between the elastotic material and the colloid-like substance, it was proposed that normal elastic fibers can change into elastotic material and then further degenerate into the colloid-like substance, eventually becoming the typical colloid substance seen in colloid milium.

Immunohistochemical and electron microscopic studies on Hashimoto's thyroiditis.

The thyroid glands of nine patients with Hashimoto's thyroiditis were studied by immunoperoxidase method for immunoglobulin (Ig) and thyroid hormone, and of these four tissue specimens were further examined by electron microscope. Immunoperoxidase method for Ig revealed that about 63% of the infiltrating cells contained Ig, and that about 90% of such Ig-containing cells had IgG. IgG-containing cells seemed to secrete autoantibodies. Immunoperoxidase method for thyroid hormone and electron microscopy revealed that there was a good correlation between the morphological features of the thyroid follicle and its immunohistochemical staining pattern. In follicles composed of columnar cells, the colloid and cytoplasm of some epithelial cells were immunohistochemically stained. Dense deposits, regarded as immune complexes, were observed in the basement membrane of these follicles, and lymphocytes were seen between the adjacent cells. When similar deposits appeared in the basement membrane of such follicles, 4 or more lymphocytes per follicle could be seen among the epithelial cells. The present findings seem to indicate that antibody-dependent cell-mediated cytotoxicity plays an important role in the pathogenesis of Hashimoto's thyroiditis.

Cervical cytology by means of fluorescence in situ hybridization with a set of chromosome-specific DNA probes.

OBJECTIVE: To reveal the numerical aberration of chromosome 1 and chromosome 17 in cervical neoplasia. METHODS: Fluorescence in situ hybridization (FISH) with chromosome-specific repetitive DNA probes was applied on cervical smears of cervical intraepithelial neoplasia (CIN) I, CIN II, and CIN III, and of invasive carcinoma cases, to detect numerical aberrations of chromosome 1 (#1) and chromosome 17 (#17). The cases were histologically classified as CIN I (n = 9), CIN II (n = 12), CIN III [severe dysplasia (n = 14), carcinoma in situ (CIS) (n = 14)], and invasive carcinoma (IC) squamous-cell carcinoma, large-cell nonkeratinizing type (n = 12). FISH was applied on the same cervical smears of these cases after Papanicolaou's staining, and copies in marked atypical cells were counted using a fluorescence microscope. RESULTS: The 9.49 +/- 2.59%/9.72 +/- 1.40% (#1/#17) cells showed an aneuploid pattern in CIN I, 22.5 +/- 3.98%/15.5 +/- 3.02% (#1/#17) in CIN II, 44.3 +/- 7.18%/ 45.01 +/- 5.61% (#1/#17) in severe dysplasia, 52.66 +/- 6.32/48.9 +/- 7.55% (#1/#17) in CIS, and 66.22 +/- 3.20%/57.38 +/- 5.35% in IC. The loss of a chromosome in CIN III cases and the gain of a chromosome in CIS and IC cases should be noted (p < 0.01). CONCLUSION: Because FISH has revealed the numerical aberrations of chromosome 1 and 17 in cervical neoplasia, this is an especially useful method for biological dosimetry and cancer biology.

Detection of numerical chromosomal aberrations in malignant melanomas using fluorescence in situ hybridization.

To evaluate the numerical chromosomal aberration in malignant melanoma, we have applied fluorescence in situ hybridization (FISH) with repetitive DNA probes specific for chromosomes 1, 6, 7, 9, 10, and 17 on 24 fresh malignant melanomas (primary: 14, metastatic: 8). We defined a tumor that had copies with more than 3 spots as chromosomal gain. Chromosomal copy number gain was found in 40.9% of the cases for chromosome 7, 27.2% for chromosome 6, 27.2% for chromosome 17, 22.7% for chromosome 9 and 10, and 4.5% for chromosome 1. Monosomy was found in 54.5% of the cases for chromosome 10, 36.5% for chromosome 9, 27.2% for chromosome 6, 22.7% for chromosome 17, and 18.1% for chromosome 1 and 7. The most frequent numerical alterations were seen in chromosomes 6, 7, 9 and 10. Gain of chromosome 6 and 7 and/or losses of chromosome 9 and 10 may play an important role in the tumorigenesis and development of malignant melanomas.

[Antiulcer properties of shosaiko-to].

Recently it has been reported that Shosaiko-to (SHO), a traditional Chinese medicine used for treating gastritis and hepatitis, also has been found useful for treating gastric ulcers, although no pharmacological study has yet investigated the precise antiulcer properties of SHO. Herein, the authors report on the results of a rat study in which the effects of SHO on gastric ulcers, acid secretions and potential difference of gastric mucosa (PD) were studied. SHO (100, 250 or 500 mg /kg, p.o.) significantly inhibited the development of ethanol-induced gastric lesions in a dose-dependent manner. SHO (500 mg/kg, p.o.) significantly inhibited the development of aspirin-,indomethacin- or water-immersion-stress induced gastric lesions. Sucralfate (500 mg/kg, p.o.) inhibited both ethanol- and aspirin-induced gastric lesions, and cimetidine (10 mg/kg, p.o.) inhibited aspirin-, indomethacin- or stress-induced gastric lesions. SHO (10, 30 and 100 mg/kg, i.p.) also significantly inhibited pentagastrin- and 2-deoxy-D-glucose (2-DG)-induced gastric acid secretions in a dose-dependent manner, whereas cimetidine (1 mg/kg, i.p.) inhibited a pentagastrin-induced secretion and atropine (0.05 mg/kg, i.p.) inhibited pentagastrin- or 2-DG-induced acid secretions. SHO (250, 500 or 1000 mg/kg, i.g.) significantly inhibited ethanol-induced PD reduction. Sucralfate (500 mg/kg, i.g.) inhibited the reduction, and cimetidine (250 mg/kg, i.g.) didn't inhibit it. These results indicate that SHO not only possesses the capability of protecting the rat gastric mucosa as well as sucralfate, but also is able to inhibit gastric acid secretions like cimetidine or atropine.

[Electron microscopic and immunohistological studies of a case of Ehlers-Danlos syndrome type IV].

A 5-year-old male considered clinically to have Ehlers-Danlos syndrome (EDS) type IV with main symptoms of fragility and easy bruisability of the skin was presented. Electron microscopic observations of collagen fibers and immunohistological examination of the localization of the type III collagen in the patient revealed dissimilarities in the size and the irregularities in the shape of collagen filaments, as well as a clear difference in localization of type III collagen when compared with normal skin of same age.

Interphase cytogenetics of melanocytic neoplasms: numerical aberrations of chromosomes can be detected in interphase nuclei using centromeric DNA probes.

This study shows that fluorescence in situ hybridization (FISH) to thin sections cut from paraffin-embedded material can be used to distinguish between groups of melanocytic neoplasms and thus may be useful as an investigational and diagnostic tool. FISH with a probe for a repeated, alpha satellite sequence specific to chromosome 17 was used to investigate the chromosomal composition of dysplastic (or Clark's nevus) and Spitz's nevi and malignant melanomas. Hybridization was to thin (approximately 6 microns) sections cut from paraffin blocks. The number of signals per nucleus in normal diploid cells is expected to be less than 2 since the sections are thinner than one nuclear diameter. Keratinocytes and lymphocytes in these same sections showed 1-2 signals per nucleus with a mean of 1.2. Dysplastic nevi showed 1-4 hybridization signals per nucleus with a mean of 1.5. Spitz's nevi showed 1-2 signals per nucleus with a mean of 1.3. Melanomas showed 1-6 signals per nucleus with a mean of 2.1. We were thus able to use FISH to demonstrate differences in chromosome numbers between groups of benign and malignant melanocytic neoplasms. Technical improvements in the near future can be expected to result in more precise estimates of chromosomal number.