Search

Objective Since most chondrocytes in articular cartilage are in the resting phase (G0) of the cell cycle, it has been difficult to investigate their cell kinetics using 3H-thymidine autoradiography, or immunohistochemistry. In the present study, DNA cytofluorometry, which is useful to analyse the cell kinetics even for such inactive cell populations as in the G0 phase, was applied to human chondrocytes of the articular cartilages under normal aging and pathologic conditions such as osteoarthritis (OA), rheumatoid arthritis (RA), and aseptic necrosis (AN). Design The human articular cartilages for the study were obtained from autopsy and surgical materials. Fifty joints were used for the study of aging, 54 for the study of OA, 20 for studying RA, and 10 for AN study. The isolated chondrocytes were quickly prepared from fresh articular cartilages, using a combination method of enzymatic digestion with papain and collagenase, followed by mechanical cell separation by churning and homogenization. Results The DNA histograms obtained by cytofluorometry with propidium-iodide staining showed that most chondrocytes had diploid DNA content (2c) in all cartilages studied, suggesting that they were in the G0 phase. However, there were a few chondrocytes having tetraploid DNA content (4c) in the normally aged articular cartilages, and there were some cells having DNA content between 2c and 4c in the diseased cartilages. The former cells were considered to be G0-phase cells of the 4c chondrocytes, while the latter cells were considered to be in the DNA synthetic (S) phase or G2-phase of the 2c chondrocytes. The frequency of 4c chondrocytes in aged cartilage was significantly increased, compared to that in the young cartilage. In contrast to the normal cartilage, the frequency of S- and G2-phase cells, which was expressed as the S– G2 index, in diseased cartilages (OA, RA and AN) was significantly high ( P 0.0001). In OA cartilage, the S–G2 index was much higher in the severe or moderate stage than in the mild stage, suggesting that the chondrocytes in clusters may actively proliferate. Conclusion These results showed that in normal articular cartilages most chondrocytes are in the G0 phase, while some became 4c polyploid cells, and that these G0-phase chondrocytes had a potential to proliferate under diseased conditions.

A recent study of multidrug resistance (MDR) 1 gene transfected osteosarcoma cells found a cause-effect relationship between increased expression of P-glycoprotein (P-gp) and a low aggressive phenotype. However, several experimental and clinical studies have observed contradictory findings in that P-gp expression has been associated with tumour progression. In the present study, we characterized P-gp-positive and P-gp-negative single-cell clones of a murine osteosarcoma, to further investigate the relationship between P-gp expression and changes in cell phenotype. Although these clones were all selected by doxorubicin (DOX) exposure, they were heterogeneous with respect to MDR1 gene expression. The P-gp-positive clones revealed MDR phenotype, whereas the P-gp-negative clones showed no resistance to drugs. Morphological and functional analysis showed that both the P-gp-positive and P-gp-negative clones were more differentiated than the parent cells in terms of enhanced activity of cellular alkaline phosphatase, an increase in well-organized actin stress fibres and enhanced osteogenic activity. Moreover, these subclones all displayed a decrease in malignant potential such as oncogenic activity, tumour growth rate and metastatic ability, regardless of their P-gp status. These results indicate that the observed osteoblastic differentiation and less aggressive phenotype in DOX-selected osteosarcoma cells may not only be explained by the direct effect of P-gp, and accordingly, consideration of the effect of DOX, as well as P-gp, appears to be important. © 2000 Cancer Research Campaign

In this study, we observed and analyzed the sub-micron motion of interphase chromosomes in living cells, labeled with the fluorescent thymidine analogue, Texas-Red dUTP. Our approach has an advantage in that chromosomes can be analyzed with regard to the nuclear architecture. We calibrated the observed motion of fluorescence-labeled chromatin by eliminating the rotational and translational movement of living nuclei that could significantly affect chromatin motion. Mathematical analyses of chromatin motion showed that: (1) interphase chromatin in living nuclei moves randomly, and the motion is limited within a small sub-region; (2) chromatin near the nuclear envelope moves in a more limited area than does centrally located chromatin; and (3) closely situated chromatin domains move independently of each other. Random and constrained chromatin motion in living nuclei supports the concept that interphase chromatin fibers are loose, flexible and floating in the nuclear matrix, and that chromatin anchors to the backbone of chromosomes. Moreover, that the random motions of DNA domains are independent of each other suggests that interphase chromatin arranges without structurally rigid continuity. This active motion of chromatin is consistent with dynamic biological processes, requiring chromosome motility and interactions. Additionally, the dynamic properties of interphase chromosomes may be significant in the interpretation of acquired chromosomal aberrations.

Overcoming multidrug resistance (MDR) is an urgent issue to improve the prognosis of osteosarcoma patients. In this study, we undertook to clarify the effect of photodynamic therapy (PDT) with acridine orange (AO) on the MDR mouse osteosarcoma (MOS / ADR1) cell line, by comparing the outcome with the effect on a chemosensitive osteosarcoma (MOS) cell line. Cultured cells of MOS and MOS / ADR1 cell lines were exposed to AO at various concentrations for various times, followed by long- or short-term (10 or 1 min) illumination with blue light (466.5 nm) for excitation. Living cells were counted by means of the trypan blue exclusion test. The results showed that AO rapidly bound to DNA, RNA and lysosomes of living MOS and MOS / ADR1 cells and also that most tumor cells in both cell lines died rapidly (viability ratio to untreated cells: 1/1000) within 48 h under conditions of continuous or 15-min flash exposure to AO at concentrations above 1.0 microg/ml plus 10-min illumination with blue light. Even after flash exposure to AO at concentrations above 1.0 microg/ml plus 1-min illumination, the viability of MOS/ADR1 cells decreased to a viability ratio of less than 1/ 1000 within 72 h. Based on these results, we concluded that AO with photo-excitation has a strong cytocidal effect, not only on chemosensitive mouse osteosarcoma cells, but also on MDR mouse osteosarcoma cells. These results suggested that photodynamic therapy with AO may be a new approach to treating MDR human osteosarcomas.

BACKGROUND. Although chemosensitivity to antiosteosarcoma agents is the most important prognostic factor in human osteosarcoma, none of the many chemosensitivity tests reported previously are reliable and clinically useful. In this study, the authors investigated the reliability and clinical availability of doxorubicin (Adriamycin; Adria Laboratories, Columbus, OH) binding assay (ABA) as a new chemosensitivity test for osteosarcoma. METHODS. Doxorubicin (adriamycin [ADM]) binding ability (%AB) to nuclear DNA in isolated osteosarcoma cells was assessed by ABA in 14 patients with primary osteosarcoma who were treated with preoperative chemotherapy containing ADM and 6 patients with relapsed osteosarcoma after intensive chemotherapy. Histologic responses to preoperative chemotherapy were evaluated by percentage of tumor necrosis (%necrosis). RESULTS. Four of the 14 patients with primary osteosarcoma had %AB > 80% (97.3 ± 3.7%) and demonstrated good histologic responses (>90% of %necrosis) to preoperative chemotherapy, whereas the remaining 10 patients had %AB < 80% (38.9 ± 21.0%) and demonstrated poor responses. Patients with recurrent osteosarcoma that was clinically evaluated to be resistant to previous chemotherapy also had low %AB (34.2 ± 28.3%). CONCLUSIONS. Because the results of the current study revealed that ABA is useful for predicting chemosensitivity to chemotherapy with ADM as well as chemotherapy without ADM for patients with osteosarcoma, and because ABA technically is simple and results can be assessed rapidly, the authors conclude that ABA is a clinically useful chemosensitivity test for patients with osteosarcoma.

To evaluate the role of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in acute respiratory distress syndrome (ARDS), we immunostained alveolar macrophages (AMs) in the bronchoalveolar lavage fluid (BALF) collected from patients with ARDS, patients in the operating room whose lung function were normal, and non-ARDS patients who were intubated and artificially ventilated for one week in the intensive care unit. We observed the significant expression of iNOS, interleukin-6 (IL-6) and interleukin-8 (IL-8), in AMs obtained from BAL in ARDS patients by the immunocytochemical approach. By comparison, statistically significant elevation was detected in IL-8 levels in BALF supernatant from the ARDS patients compared with the control and the non-ARDS patients. These results suggest that the expression of proinflammatory cytokines and iNOS in AMs as determined by immunocytochemical technique may be an important predictor of the early stage of lung injury in ARDS. We suggest that NO and proinflammatory cytokines produced by AMs might play a pivotal role in acute lung injury. The immunocytochemical approach could be the most sensitive predictor for activated AMs in ARDS patients.

Recently, interphase cytogenetics using fluorescent in situ hybridization (FISH) was performed on various kinds of solid tumor, and their inherent karyotypic heterogeneities were revealed. Concerning this heterogeneity, we evaluated both the exhibiting number of chromosome 17 and nuclear DNA content on an identical nucleus by means of computer-controlled auto-scanning stage in order to demonstrate the alteration in number of chromosome 17 among cytofluorometrically distinct subpopulations.We investigated 8 lesions of surgically resected colorectal carcinomas, which were classified as aneuploid in quantitative DNA analysis and also exhibited an increase of 17-aneusomy nuclei. We used paraffin-embedded archival blocks. First, we prepared isolated cell specimens, and memorized the position of the cells on a glass slide using computer-controlled auto-scanning stage. Next, the specimens were stained with propidium iodide, and the fluorescent intensity was evaluated as nuclear DNA content in the order of cell position data. And lastly, FISH was performed with (peri) centromerespecific DNA probes for chromosome 17, and we enumerated the number of signals in a nucleus also according to cell position data. Then, we compared the distribution of number of chromosome 17 among cytofluorometrically distinct subpopulations. Three of 8 lesions showed a single GO+G1 peak, and the rest exhibited plural GO+G1 peaks in DNA profile. And 4 of 5 lesions, which showed plural GO+G1 peaks, presented a peak at the DNA value of (near) 2c. We could detect an alteration in the distribution of number of chromosome 17 between diploid peak and aneuploid peaks in 4 of 4 lesions which presented a peak at the DNA value of (near) 2c. However, we could ot find a difference in the distribution of number of chromosome 17 between GO+G1 peak and G2+M peak. These observations indicate that the distribution of number of chromosome 17 reflects an endoreduplication of genome content, yet, it does not alter in accordance with the phase of cell cycle. It is necessary to evaluate nuclear DNA content simultaneously in order to assess an essential cytogenetic change.

私たちの教室で治療した胃癌症例中で多発胃癌は5.6%あるが, 早期胃癌の中では多発のものは8.6%を占めて近年増加傾向にある. この多発早期胃癌は単発早期胃癌と比較して高齢者に多く, 隆起型, 分化型 (高分化腺癌) の頻度が高く, 占居部位は大半が胃体部, 幽門部で幽門側切除が可能であった. 私たちは多発早期胃癌の悪性度評価法として, 各病巣の癌細胞増殖動態を顕微測光法で解析してきた. 予後良, 好例では各病巣のDNAプロイディ・パターンはdiploidで増殖動態は類似し, 病巣間で著差を認めなかった. 一方, 予後不良例ではS-G2期細胞の増加, 多倍体化などの進行胃癌で見られる増殖動態を示し, これらの所見は病巣間でばらつきを示した. したがって, DNAプロイディ・パターンは多発早期胃癌の悪性度の指標になりうると考えられた. また, リンパ節転移の頻度は単発例と差がなく, 単発早期胃癌と同様な進行度評価を適用しうると考えられた.

To elucidate the role of proliferating cell nuclear antigen (PCNA) in vivo, PCNA immunohistochemistry combined with 3H-thymidine autoradiography was performed on 2 types of regenerating rat livers. One group of rats underwent 2/3 partial hepatectomy (PH), and the other group 1/3 PH. After 3H-thymidine labeling, the livers were fixed with paraformaldehyde (PFA) and methanol, and paraffin-embedded. In methanol-fixed tissues, the serial changes of the PCNA-positive index (PCNA-PI) corresponded well with those of the 3H-thymidine-labeling index (3H-thymidine-LI) after each PH. In PFA-fixed tissues, however, the PCNA-PI was 26% before PH, when the 3H-thymidine-LI was below 1%. Furthermore, the 1/3 PH group, despite a continued low 3H-thymidine-LI, showed a rise in PCNA-PI to a level comparable to that in the 2/3 PH group. From these results, we propose 3 phases of PCNA expression. In PFA-fixed tissues, PCNA that was detectable before PH was considered pooled in G0-phase nucleoplasm (Phase I), and some or many of the PCNA-positive cells failed to enter the S-phase in the 1/3 PH group, reflecting the increased PCNA not bound to DNA replicons (Phase II). In methanol-fixed tissues, PCNA was detected only in the S-phase, indicating its direct involvement in DNA synthesis (Phase III). Therefore, determining PCNA-PI in methanol-fixed tissues is useful to evaluate proliferative activity, whereas that in PFA-fixed tissues must be assessed with great caution.

症例は67歳女性.呼吸困難,乾性咳,発熱にて入院した.現病歴,胸部X線写真, CTなどより特発性間質性肺炎と診断した.抗生物質,ステロイドの投与にていったん症状の改善を認めたが,入院中再び呼吸困難が増悪し,ステロイドの増量にもかかわらず呼吸不全にて死亡した.剖検では肺炎所見を認めず,死因は急速に進行した肺線維化による呼吸不全と右心不全によるものと考えられた.経過中,補体分解産物iC3, Bb, c4dおよびC3a, C5aを経時的に測定できた.剖検所見と補体の動きからその病態に補体の活性化がかかわっていたことが示唆された.

Bromodeoxyuridine (BrdU) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. To determine the optimal conditions for detecting BrdU-incorporated nuclear DNA in formalin- or ethanol-fixed, paraffin-embedded human gastric tissues, we tested several different pretreatment procedures of hydrolysis with HCl and enzymatic digestion with actinase before the immunohistochemical staining. Three cases of surgically resected stomachs were perfused with artificial blood containing BrdU and fixed in formalin, ethanol or with their combination for different durations. The results of BrdU-immunohistochemical stainings were checked and the labeling indices were compared. As for hydrolysis, treatment with 2N HCl at 25°C for 90min was sufficient to detect the immunoreactivity in the stomach irrespective of fixative conditions tested. However, to obtain the most suitable stainings an additional enzymatic digestion was needed, and the optimal condition with actinase was different according to the fixation time in formalin- and/or ethanol-fixed tissues. There are also a few differences in the demonstrability of nuclear BrdU among various kinds of cells, i.e. normal epithelial cells, immunoblasts in the lymphoid follicle and cancer cells. Our method could extend the range of application of BrdU immunohistochemistry for cell kinetic studies using human surgically resected organs.

私達の近年の開発研究に基づく顕微測光と画像解析の結合解析法を応用し, 顕微蛍光測光法による細胞増殖動態の解析単独では鑑別の困難な, 甲状腺の腺腫様過形成5例濾胞腺腫12例および濾胞癌12例を検索した.単離採取した一つひとつの細胞について, 細胞測光による核DNA定量と細胞核の形態解析 (面積, 周囲長, 核DNA濃度, 形状係数など) を対応させて行った.各症例について細胞周期の全ての時期の細胞 (全細胞) 群に対して形態解析を行うとともに, 細胞周期に基づく形状変化の影響を除外して客観的に形態学的特性を評価するために, 2倍体細胞 (G0-G1期細胞) 群に限定した解析も行った.その結果, 検索した腺腫様過形成, 濾胞腺腫および濾胞癌の核DNA量分布パタンは類似していたが, 2倍体細胞の核形態の不規則さは, 腺腫様過形成や腺腫よりも腺癌において有意に高度であることが定量的に示された.以上より, 本法が, 甲状腺腫瘍の良・悪性鑑別に, 細胞測光法や画像解析法単独による解析よりも, 有用であると考えられた.

The modified Bodian method with protargol (silver protein) is ordinarily used to detect nerve fibers. With this technique, applied to decalcified rat bone sections, the bone canaliculi were clearly stained black with good contrast to the bone matrix in both lamellar and woven bone. In addition, the connections between the bone canaliculi and other canaliculi, osteoblasts, osteoclasts, and chondrocytes were easily detectable. We found that the bone canaliculi of woven bone were fewer in number and ran more irregularly than those of lamellar bone. We believe that this staining method for bone canaliculi in decalcified bone is superior to previously reported methods and may be useful in studies on bone pathology.

Objective Although it is well known that binuclear cells commonly appear among the chondrocytes of normal cartilages as well as among neoplastic chondrocytes of chondrosarcomas, the mechanism of binucleation is still unclear. Therefore, this study was undertaken to clarify the mechanism of binucleation in chondrocytes, using primary culture cells of growth plate cartilage. Design These chondrocytes were exposed to acridine orange (AO) which is a fluorescent dye for differentiating certain DNAs and RNAs in nuclei and cytoplasm, and which inhibits mitosis. After exposure to 0.5μg/ml AO, for 0, 6, 24, 48, and 96h, the following parameters were investigated: (1) cell growth rate (GR); (2) frequency of hyperdiploid cells (%HDC) by DNA cytofluorometry; (3) mitotic index (MI); (4) BrdU labeling index (LI); (5) frequency of binuclear cells (%BNC). Results Compared with the control cells, which were cultured in AO-free medium, the GR was remarkably inhibited at 24h. MI was also decreased from 6 to 24h, and LI decreased at 48h. However, these parameters were recovered at 96h. The %HDC was increased from 6 to 96h, and the %BNC was also increased to a maximum of six times that of the control cells at 96h. Discussion These results suggested that the binuclear cells observed among the cultured chondrocytes may be formed from G2 arrested cells by amitotic nuclear division, but not by mitosis without cytoplasmic division or cell fusion.

We recently found that silver impregnation staining with protargol (silver protein), that is, a modified Bodian method, is useful for histologically identifying the details of bone canaliculi structure, using thin sections of decalcified bone tissues. With this staining method, we conducted the present study to assess the development of bone canaliculi during the process of intramembranous ossification using a fracture-like stimulation model of the rat femur. After making a drill-hole in the cortex of the rat femur, decalcified thin sections were obtained after 3, 5, 7, and 14 days by the standard paraffin-embedding procedure. Silver staining for bone canaliculi was performed using our previously reported technique. The results showed that woven bone covered the fracture surface of the cortex after 5 days, then immature lamellar bone attached to the woven bone after 7 days, and finally the lamellar bone matured and became thick with appositional growth after 14 days. The osteocytes in the woven bone appeared at an early stage of bone repair and developed a few canaliculi that were short and irregularly distributed in the osteoid matrix, while the osteocytes in the lamellar bone at a late stage formed many bone canaliculi that were long and regularly distributed in mature bone matrix. Therefore, we concluded that woven bone osteocytes may be necessary for induction of the lamellar bone osteocytes followed by active appositional growth of the lamellar bone at the early stage of bone repair, and also that both bone tissues could be clearly distinguished from one another based on the pattern of development of bone canaliculi by the osteocytes, as seen with the use of our sensitive staining method.

PURPOSE: The efficacy of the reduction time (tau) after castration as a prognostic factor was examined by comparing to Gleason score. MATERIALS AND METHODS: The change of prostatic volume after castration was observed from the castration to 3 months after in 24 cases of prostatic cancer. Prostatic volume was examined by transrectal ultrasonography of the prostate. Survival curves was calculated by Kaplan-Meier method. Differences among survival curves were analyzed using Cox-Mantel test. RESULTS: tau had a close relationship to the prognosis of each case (Wilcoxon test: p < 0.05, Cox-Mantel test: p < 0.05). Gleason score had a weak relationship to prognosis (Wilcoxon test: N. S., Cox-Mantel test: p < 0.05). CONCLUSIONS: tau was efficient as prognostic factor compared to Gleason score.

Whether spermatocytic seminoma (SS) develops during meiosis or not is still controversial. To determine that, the DNA content of large, small, and intermediate cells of SS was measured by cytofluorometry to obtain DNA histograms for each cell type. The large cells were aneuploid and showed cell cycles with G0/G1 DNA values from 3C to 18C. The ploidy of the large cells doubled stepwise (i.e., 4.5C-9C-18C, 3C-6C-12C, and so forth). The small cells showed distinct cell cycles with a DNA content of 2C or near 2C, and the intermediate cells showed both diploid and aneuploid DNA values. It is speculated that none of the cell types is involved in meiosis, based on the fact that each size of cell appears to have its own cell division cycle. It is concluded that in SS, the diploid cell cycle formation by small cells and the creation of large cells by continuous sequential polyploidization result in the morphologic expression of three types of cells (large, small, intermediate).

An autostage cytofluorometry system for multiparametric analysis is described. The system consists of a compact epi-illumination cytofluorometer equipped with an autostage device, a small data processing unit, an autostage controller and a microcomputer. In this system, repeated cytofluorometry and morphological observations for the same series of cells can be made at predetermined cell positions (X, Y-values) on a slideglass, permitting accurate multiparametric cytofluorometry in the same cells, even for a mixed cell population. Fluorochromes can be applied in any combination, if they can be utilized sequentially by staining and destaining cells. Application experiments demonstrated that the present system allows detailed cell cycle analysis.

症例は29歳女性.上腹部痛を主訴として,昭和59年8月2日入院.血液学的検査で,中等度の貧血と血小板の減少,血清LDHの軽度上昇を認めた.出血時間,プロトロンビン時間共に延長していた.AFPは陰性.腹部血管造影では,肝・脾血管腫と診断され,さらに骨転移巣も認めた為,血管肉腫が強く示唆された.確定診断の目的で腹腔鏡下肝生検を試みたが,出血が激しく断念し,外科的肝切除にて血管肉腫と診断された.本例は,その後肝機能が悪化し,昭和60年3月22日に肝不全で死亡した.剖検では,肝臓,脾臓,骨髄に血管肉腫を認めたが,原発巣は不明であった.肝血管肉腫は生前診断が困難であり,特に肝血管腫や肝嚢胞性病変との鑑別が問題となる.疑いのある症例には,速やかに開腹下肝生検,あるいは肝切除術を行うことが必要と考えられた.

Using in vivo local labeling method, cell proliferation and differentiatiation in the human epidermis covering the mammary gland of 41 and 42 year old women were studied by means of 3H-thymidine autoradiography.1. Stratum germinativum or G-zone of the human epidermis in which cells are proliferating corresponds to epibasal and basal layers, but not the basal layer alone. Proliferating cells are predominantly located in the epibasal layer.2. Melanin containing cell in the basal layer is a kind of minor-differentiated cell and proliferates at much slower rate than the non-pigmented basal cell.3. Generation time of the genertive cell is estimated at 4 days and the maximum length of DNA synthesis, at 10 hours. Many differentiating cells stay in the epibasal layer and they amount to 50% of the total cells in the layer. Their transit time in the layer is estimated at an average of 4 days.4. The unequal division or (G, M) division is not a mechanism to maintain the steady state, nor do any types of mitosis play a decisive role in the regulation of cell differentiation. Mitoses yield two equivalent daughter cells which are in the same state as the mother cell.5. Cell differentiation in the human epidermis seems to be determined by the rule of probability in an individual daughter cell during t1 or G1 period, as has been confirmed in various other cell systems.

血管腫を伴ったリンパ管腫,いわゆるhemolymphangiomaは,ほとんど報告をみない.最近,我々は後腹膜原発のhemolymphangiomaを経験し,超音波, CT,血管造影などに加え,磁気共鳴映像法(MRI)を施行し,術前診断を試みた。嚢腫内容は, CTではenhanceされなかったが, MRI計算値においてT1時間の著明な短縮を示したことから陳旧性血液成分と診断でき,内容の性状診断にMRIは有用であった.また,術後の免疫組織学的検索では,血管腫の内皮だけでなく,リンパ管腫の内皮にも,第VIII因子関連抗原が陽性であった.既報告の後腹膜リンパ管腫99例とhemolymphangiomaの病態を比較し,リンパ管腫の発生に関し,若干の考察を加えて報告する.

To obtain good chromosome banding with “33258 Hoechst,” various conditions were examined. For fluorescence microscopy, an UV filter for excitaion and a BV filter for emission were selected from spectrofluorometric examination. “33258 Hoechst” was dissolved in distilled water and good chromosome banding was obtained when slides were stained at room temperature, for 10 to 20 minutes at the concentration between 4 μg/ml to 0.2 μg/ml of this dye. The aging of slides before and after staining was shown to be another important factor. Chromosome banding revealed with “33258 Hoechst” represents chromosome banding corresponding to both Q-and C-bandings in a very simple way. The mechanism of chromosome banding with “33258 Hoechst” is briefly discussed.