Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts.

Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase-immortalized normal human fibroblast cell line TIG-1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near-tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase-immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc.

Establishment of proliferative tetraploid cells from normal human fibroblasts.

The chromosomal instability of polyploid cells, which leads to the formation of aneuploid cells, is causally related to carcinogenesis in human tissues. However, the precise link between the chromosomal instability of polyploid cells and oncogenic transformation of them remains elusive. This is partly because we lack an experimental model in which non-transformed polyploid human cells can propagate in vitro. In a previous report, we demonstrated that proliferative tetraploid cells can be established from TIG-1 human fibroblasts by treatment with the spindle poison demecolcine (DC, colcemid) for 4 days. However, this procedure could not be applied to other human fibroblast strains because the resulting cells proliferated as a mixture of diploid and tetraploid populations. Here, we report a modified procedure to establish proliferative tetraploid cells from human fibroblasts of the BJ strain with minimum contamination by diploid cells. In the modified procedure, DC-arrested mitotic cells were collected by mitotic shake-off and treated with DC for an additional 3 days. DC-treated cells restarted proliferation as tetraploid cells after several days of growth arrest and showed similar growth to that of untreated diploid cells. The MDM2 antagonist Nutlin-3a activated p53 in established tetraploid cells and suppressed their growth, indicating that these cells have functional p53. These results contradicted the hypothesis that p53 functions as the tetraploidy checkpoint and prevents proliferation of tetraploid cells. Tetraploid cells established by our method could be a valuable model for the study of chromosomal instability and the oncogenic potential of polyploid cells.

Formation of bipolar spindles with two centrosomes in tetraploid cells established from normal human fibroblasts.

Tetraploid cells with unstable chromosomes frequently arise as an early step in tumorigenesis and lead to the formation of aneuploid cells. The mechanisms responsible for the chromosome instability of polyploid cells are not fully understood, although the supernumerary centrosomes in polyploid cells have been considered the major cause of chromosomal instability. The aim of this study was to examine the integrity of mitotic spindles and centrosomes in proliferative polyploid cells established from normal human fibroblasts. TIG-1 human fibroblasts were treated with demecolcine (DC) for 4 days to induce polyploidy, and the change in DNA content was monitored. Localization of centrosomes and mitotic spindles in polyploid mitotic cells was examined by immunohistochemistry and laser scanning cytometry. TIG-1 cells treated with DC became almost completely tetraploid at 2 weeks after treatment and grew at the same rate as untreated diploid cells. Most mitotic cells with 8C DNA content had only two centrosomes with bipolar spindles in established tetraploid cells, although they had four or more centrosomes with multipolar spindles at 3 days after DC treatment. The frequency of aneuploid cells increased as established tetraploid cells were propagated. These results indicate that tetraploid cells that form bipolar spindles with two centrosomes in mitosis can proliferate as diploid cells. These cells may serve as a useful model for studying the chromosome instability of polyploid cells.

Significant Correlation between Chromosomal Aberration and Nuclear Morphology in Urothelial Carcinoma.

We aimed to identify whether there is any correlation between chromosomal/genetic changes, nuclear morphology and the histological grade of urothelial carcinomas of the urinary bladder. Morphometry and multicolour fluorescence in situ hybridisation (FISH) techniques were applied to 250 cells in five low-grade cases and 350 cells in seven high-grade cases of urothelial carcinoma. Compared with low-grade carcinomas, most high-grade cases showed larger and more variable nuclear size, more frequent polysomy of centromere enumeration probes (CEPs) 3, 7 and 17, and the loss of the 9p21 locus. The number of CEP signals in cells was increased as the nuclear area of the cells became larger. Cells with gains in two or more types of CEP had significantly larger nuclei than cells with normal FISH signal patterns. In conclusion, the present study indicates that there was a correlation between nuclear morphology and chromosomal/genetic changes which were related to histological grading. Thus, we show that differences in the chromosomal/genetic aberrations present in low- and high-grade tumours can affect not only nuclear morphology but also the histopathological and clinical behaviour of urothelial carcinomas.

Cellular aging and centrosome aberrations.

The significant increase in chromosomal instability with aging is well known, but the underlying mechanism is not fully understood. Our earlier studies showed a high frequency of abnormal mitosis, such as mitotic slippage or incomplete mitosis in near-senescent human fibroblasts. This study examined the centrosome aberrations in mitotic and interphase cells from different passages of several strains of human fibroblasts. Analysis by laser scanning cytometry showed increased frequencies of abnormal mitotic cells with supernumerary (>2/cell) centrosomes and misaligned chromosomes in later passages in all strains examined. Numerical centrosome aberrations were prominent in a polyploid subpopulation. In metaphase cells, numerical centrosome aberrations were correlated significantly with chromosome misalignment. Fluorescent in situ hybridization analysis using a centromere-specific probe revealed a correlation between chromosome aneusomy and centrosome over-duplication. These results suggest that abnormal duplication of centrosomes in association with cellular aging may be responsible for the increase in chromosomal instability with aging.

A molecular analysis of biclonal follicular lymphoma: further evidence for bone marrow origin and clonal selection.

We report a follicular lymphoma (FL) case presenting the coexistence of two tumor cell subpopulations in lymph node (LN) and bone marrow (BM), which exhibited an inverse pattern of immunoglobulin light (IgL) chain gene rearrangement and expression: Igkappa-lambda+ in LN and Igkappa+lambda- in BM. These tumor clones shared an identical BCL2-IgH recombination, accompanying t(14;18)(q32;q21) translocation, and an identical variable, diversity and joining segments joining with clone-specific VH somatic hypermutations on the untranslocated IgH allele. Our study provides further evidence that FL clones, originating from common progenitor cells, can be developed independently at different sites and with different IgL expression after immune selection.

Laparoscopic cecopexy for mobile cecum syndrome manifesting as cecal volvulus: report of a case.

A 44-year-old woman was admitted to our hospital for investigation and treatment of sudden abdominal pain and distention. Plain abdominal radiography and abdominal computed tomography (CT) findings were suggestive of sigmoid volvulus. She underwent an emergency colonoscopy, and the scope passed easily through the sigmoid colon and reached the ascending colon quickly. However, stenosis with concentricity of the fold was observed in the cecum, which was shifted upward and to the left. Based on these findings, we diagnosed cecal volvulus caused by mobile cecum syndrome. The patient's symptoms resolved quickly after colonoscopic reduction and elective laparoscopic surgery was performed 18 days after admission. Perioperative examination revealed a mobile cecum caused by an elongated ascending colon. We sutured the cecum and ascending colon to the lateral peritoneum laparoscopically with interrupted sutures. The patient recovered well and was discharged on postoperative day 7. An unfixed intestine can be detected easily during laparoscopic surgery, which is minimally invasive and cosmetically, physically, and economically beneficial. Thus, we recommend laparoscopic cecopexy for mobile cecum syndrome.

Early laparoscopic cholecystectomy for acute gangrenous cholecystitis.

Treatment of severe acute cholecystitis by laparoscopic cholecystectomy remains controversial because of technical difficulties and high rates of complications and conversion to open cholecystectomy. We investigated whether early laparoscopic cholecystectomy is appropriate for acute gangrenous cholecystitis. Pathologic diagnoses and outcomes were analyzed in patients who underwent laparoscopic or open cholecystectomy at our hospital, January 2002 to September 2005. Of 30 patients with acute gangrenous cholecystitis, 16 underwent early laparoscopic cholecystectomy, 10 underwent open cholecystectomy, and 4 were converted to open cholecystectomy (conversion rate, 20.0%). There was no significant difference in operation time or intraoperative bleeding. The requirement for postoperative analgesics was significantly lower (6.4+/-7.3 vs. 1.5+/-1.2 doses, P<0.05) and hospital stay significantly shorter (8.6+/-2.1 vs. 15.6+/-6.3 d, P<0.01) after laparoscopic cholecystectomy. There were no postoperative complications in either group. Thus, early laparoscopic cholecystectomy seems appropriate for acute gangrenous cholecystitis. Conversion to open cholecystectomy may be required in difficult cases with complications.

Laparoscopic resection of an ileal lipoma: Report of a case.

A 63-year-old woman was admitted to our hospital for investigation of upper abdominal pain and vomiting. Ultrasonography (US) showed a hyperechoic mass in the right lower abdomen, and computed tomography (CT) showed a low-density mass and intestinal invagination. Thus, we made a diagnosis of intestinal lipoma with intussusception and performed laparoscopic partial resection of the ileum, including the tumor. The resected specimen contained a round tumor, 25 x 22 x 20 mm, which was identified as an intestinal lipoma histopathologically. Our experience supports earlier reports that US and CT are effective tools in the diagnosis of bowel lipoma. Laparoscopic surgery is the treatment of choice for benign tumors of the small intestine because it is minimally invasive, with cosmetic, physical, and economic benefits.

Neovascularization induced by autologous bone marrow cell implantation in peripheral arterial disease.

Neovascularization has recently been used as a new treatment for severe ischemic disease. We tried to induce therapeutic neovascularization by autologous bone marrow cell implantation (BMCI) in eight selected patients with chronic peripheral arterial disease (PAD), in whom traditional treatments had failed. Improvement of subjective symptoms was seen in seven patients after treatment. Of three limbs with toe or finger ulceration, complete healing was achieved in two, while the other one became less severe after treatment. No relative toxicity was observed in any of the patients. BMCI might be a feasible treatment for selected patients with chronic PAD.