Myeloperoxidases of human myeloid leukemia cells HL-60 grown in culture and in nude mice

Human myeloid leukemia HL-60 cells were grown either in suspension culture or in nude mice. The two types of myeloperoxidase, the small and the large type, in crude extracts of these cells were analyzed by sucrose density gradient centrifugation. The proportions of the small and the large myeloperoxidase varied markedly depending on the growth conditions of cells. In cells in culture, the small myeloperoxidase amounted to 80% of the total myeloperoxidase, whereas in the solid tumors it amounted to only about 30% of the total. Both in cultured cells and solid tumors, 40% of the total myeloperoxidase was found in the soluble fraction and the rest in the granule fraction. However, adult and fetal blood granulocytes contained only the large myeloperoxidase, which was mainly recovered in the granule fraction. Antiserum prepared against purified large myeloperoxidase of HL-60 solid tumors reacted with the small myeloperoxidase as well as the large enzyme of HL-60 cells in culture. The antiserum also precipitated myeloperoxidase of adult and fetal blood granulocytes. An Ouchterlony double immunodiffusion test also revealed their immunological identity.     

Characteristics of rat normal mammary epithelial cells and dimethylbenzanthracene-induced mammary adenocarcinoma cells grown in monolayer culture

Summary The characteristics of normal mammary epithelial and 7,12-dimethylbenz[a]anthracene (DMBA)-induced adenocarcinoma cells derived from rats and grown in monolayer culture were compared. Normal mammary epithelial cells exhibited different morphology and agglutinability by plant lectins, slower growth rate, and lower saturation density and cloning efficiency. In addition, the normal cells were sensitive to the toxic effect of DMBA, and were unable to grow in soft agar or to form tumors, when inoculated into newborn Sparague-Dawley rats. The converse was true in each case for the adenocarcinoma cells. Supported by Public Health Service Research Grant CA 01237603 from the National Cancer Institute Portions of this paper were presented at the 65th Annual Meeting of the American Association for Cancer Research at Houston, Texas, 1974.     

Assessing the tumorigenic phenotype of VERO cells in adult and newborn nude mice.

VERO cell lines are important substrates for viral vaccine manufacture. The mechanism by which these cells became neoplastically transformed is unknown. During tissue-culture passage, VERO cells can develop the capacity to form tumors. Although at the passage levels (around p140) currently used for vaccine manufacture, VERO cells are non-tumorigenic, questions have been raised about safety issues that might be associated with this capacity to acquire a tumorigenic phenotype. To begin to address these issues, the tumorigenicity of VERO cell lines, derived at different passage levels under different growth conditions, were evaluated in 365-day assays in adult and newborn nude mice. High passage (p>200) VERO cell lines established by random passaging in tissue culture produced tumors in adult (10 out of 27) mice and newborn (21 out of 30) mice, respectively. In contrast, a high passage (p>250) cell line established by passage at sub-confluence produced tumors only in newborn mice (16 out of 30). Progressively growing tumors began forming at 36 days in newborns and at 69 days in adults. Higher tumor incidences and shorter tumor latencies suggest that newborn nude mice may be more sensitive than adults in detecting the expression of a tumorigenic phenotype by some VERO cell lines.     

Long-term culture of normal human colonic epithelial cells in vitro.

Studies of normal cellular function as well as the understanding of cellular mechanisms of carcinogenesis and other diseases of the large intestine have been limited, particularly due to the lack of long-term culture of normal human large intestinal epithelial cells (NHLIEC). Using the epithelia from surgically resected human colon, we have dissociated a sufficient number of viable NHLIEC and maintained them in in vitro culture for up to 5 months. Normal-appearing human large intestinal mucosal fragments (1 mm2) were treated with 0.01 mg/ml trypsin, 0.2 mg/ml collagenase + 0.1 mM EGTA or 0.1 mg/ml trypsin + 0.1 mM EGTA in a Stomacher laboratory blender to isolate the cells. Compared with other methods, the use of the Stomacher blender combined with low concentrations of proteolytic enzymes yielded greater numbers of cells per gram of tissue, with up to 84% viable cells. Primary and serially passaged NHLIEC were cultured in CMRL-1066, MEM with 5% serum, and serum-free KGM. These media were all supplemented with insulin, hydrocortisone, epithelial growth factor, and bovine pituitary extract. CMRL-1066 was found to be the best medium for NHLIEC. Contaminating fibroblasts were selectively removed by briefly allowing the cells to adhere to the culture vessel and adding 25 U/ml collagenase to the culture media at the first subculture treatment. The epithelial nature and secretory function of the established cells were confirmed by morphological criteria (light microscopy, phase contrast microscopy and electron microscopy), immunoreactivity to cytokeratin, and positive mucin cytochemistry. We propose that using this methodology for the culture and maintenance of NHLIEC for an extended period of time would serve as a valuable model for a variety of investigations.     

Amiodarone induces apoptosis of human and rat alveolar epithelial cells in vitro

The antiarrhythmic amiodarone (AM) and its metabolite desethylamiodarone (Des) are known to cause AM-induced pulmonary toxicity, but the mechanisms underlying this disorder remain unclear. We hypothesized that AM might cause AM-induced pulmonary toxicity in part through the induction of apoptosis or necrosis in alveolar epithelial cells (AECs). Two models of type II pneumocytes, the human AEC-derived A549 cell line and primary AECs isolated from adult Wistar rats, were incubated with AM or Des for 20 h. Apoptotic cells were determined by morphological assessment of nuclear fragmentation with propidium iodide on ethanol-fixed cells. Necrotic cells were quantitated by loss of dye exclusion. Both AM and Des caused dose-dependent necrosis starting at 2.5 and 0.1 microg/ml, respectively, in primary rat AECs and at 10 and 5 microg/ml in subconfluent A549 cells (P < 0.05 and P < 0.01, respectively). AM and Des also induced dose-dependent apoptosis beginning at 2.5 microg/ml in the primary AECs (P < 0.05 for both compounds) and at 10 and 5 microg/ml, respectively, in the A549 cell line (P < 0.01). The two compounds also caused significant net cell loss (up to 80% over 20 h of incubation) by either cell type at drug concentrations near or below the therapeutic serum concentration for AM. The cell loss was not due to detachment but was blocked by the broad-spectrum caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Furthermore, the angiotensin-converting enzyme inhibitor captopril (500 ng/ml) and the angiotensin-receptor antagonist saralasin (50 microg/ml) significantly inhibited both the induction of apoptosis and net cell loss in response to AM. These results are consistent with recent work from this laboratory demonstrating potent inhibition of apoptosis in human AECs by captopril (Uhal BD, Gidea C, Bargout R, Bifero A, Ibarra-Sunga O, Papp M, Flynn K, and Filippatos G. Am J Physiol Lung Cell Mol Physiol 275: L1013-L1017, 1998). They also suggested that the accumulation of AM and/or its primary metabolite Des in lung tissue may induce cytotoxicity of AECs that might be inhibitable by angiotensin-converting enzyme inhibitors or other antagonists of the renin-angiotensin system.     

Polyplex-mediated gene transfer into human retinal pigment epithelial cells in vitro

The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age-related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome-mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex-mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex-mediated gene transfer, but stable integration does occur at low frequencies with and without selection.     

Decrease in adherence of bacteria and yeasts to human mucosal epithelial cells by noxythiolin in vitro

Adherence of buccal and vaginal isolates of Candida albicans to buccal epithelial cells and the adherence of urine isolates of Escherichia coli and Staphylococcus saprophyticus to uroepithelial cells was quantified by light microscopy. The antimicrobial agent noxythiolin reduced the adherence of these micro-organisms in both exponential and stationary growth phases. Adherence of both the blastospore and pseudohyphal forms of C. albicans was reduced. Treatment of epithelial cells and/or micro-organisms with noxythiolin resulted in decreased adherence. No anti-adherence effect was observed with formaldehyde and N-methylthiourea, the degradative products of noxythiolin.     

Desmoplakin expression and distribution in cultured rat bladder epithelial cells of varying tumorigenic potential

The expression and distribution of the desmosomal plaque proteins, desmoplakins (DPs) I and II, were studied in nontumorigenic (RBE-8) and a series of tumorigenic (AY34, R-4909, SS-24B, RBTCC-8, and 804G) rat bladder epithelial cell lines. These cell lines ranged from slow-growing papillary transitional cells (AY34) to rapidly metastatic carcinoma cells (RBTCC-8). DPs I and II were shown by immunoblotting and Northern analysis to be present in nontumorigenic RBE-8 cells as well as in all of the tumorigenic cell lines, albeit in differing amounts. Immunofluorescence microscopy revealed striking differences in DP distribution, corresponding in general with increases in tumorigenic potential. Whereas DPs of normal RBE-8 cells and less tumorigenic AY34 cells were localized predominantly at cell interfaces, the more tumorigenic lines exhibited a high proportion of DP in the form of cytoplasmic dots, a distribution reminiscent of that seen in epithelial cells maintained in low levels of extracellular calcium. In 804G cells, which represented the most extreme example of this phenomenon, the majority of DPs were organized as cytoplasmic dots. Electron microscopy revealed intermediate filament (IF)-associated spots in the cytoplasm as well as an elaborate array of IF-associated plaques at the cell-substratum interface. The IF-associated spots in the cytoplasm reacted with anti-DP antibody in immunogold labeling experiments while those at the cell-substratum did not react. In more dense cultures of 804G cells, certain cells stratified and expressed increased amounts of DP followed by the induction of new keratins including those of the skin type. Decreasing extracellular calcium resulted in a rearrangement of DP in each cell line; staining at cell-cell interfaces disappeared and was replaced with a pattern of cytoplasmic dots. These results demonstrate a possible relationship between desmosome assembly and/or maintenance and tumorigenic potential.     

Secretion of a TGF-beta-like growth inhibitor by normal rat mammary epithelial cells in vitro

We have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures. RME cell-derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration. Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity. The acid-activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio-Gel p60 column with a peak of activity in the 28 kDa range. Incubation of the acidified-concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)-beta completely abolished the inhibitory activity of the CM. Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF-beta antiserum was greater than that observed in growth medium alone. Subsequent experiments demonstrated that addition of TGF-beta antiserum alone to serum-free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25-fold higher concentrations. Zymographic analysis of RME-CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor. These results indicate that normal RME cells secrete a latent TGF-beta-like growth factor into conditioned medium. Furthermore, the results indicate that some of the latent growth factor is activated in situ and contributes to the growth potential of the cells in primary culture in an autocrine manner.     

Transformed human bronchial epithelial cells (beas-2b) alter the growth and morphology of normal human bronchial epithelial cells in vitro

Normal human bronchial epithelial cells (BE) and adenovirus-12 SV40 hybrid virus transformed, non-tumorigenic human bronchial epithelial cells (BEAS-2B) were cultured for 7 days in a serum free hormone supplemented medium. BE cells after 3 days in culture were exposed to conditioned medium (CM_t) from confluent BEAS-2B cells. By day 7, CM_t-treated BE cells exhibited a lower colony forming efficiency (CFE), fewer cells per colony, and a reduced mitotic index (MI) and BrdU (bromodeoxyuridine) labeling index. CM_t also enhanced the expression of a terminally differentiated squamous phenotype in BE cells. Cell free lysates from BEAS-2B cells (CFL_t) had effects similar to CM_t on the MI and morphology of BE cells. In contrast, CM_t and CFL_t did not inhibit the growth, or alter the morphology of BEAS-2B cells. Conditioned medium from BE cells (CM_n) did not reduce the growth of BEAS-2B cells, and had little effect on the morphology of BE cells. In co-culturesAbbreviations BE normal bronchial epithelial cells - BEAS-2B adenovirus-12 SV40 hybrid virus transformed bronchial epithelial cells - CM_n conditioned medium from BE cells - CM_t conditioned medium from BEAS-2B cells - CF_n cell free lysate from BE cells - CFL_t cell free lysate from BEAS-2B cells - BrdU bromodeoxyuridine - KGM keratinocyte growth medium - TGF- transforming growth factor type - NCI-LHC National Cancer Institute-Laboratory of Human Carcinogenesis Contribution No. 2801 from the Pathobiology Laboratory, University of Maryland.     

Cell proliferation kinetics in two human tumors grown in athymic nude mice.

The technique of labelled mitoses was used to investigate the cell proliferation kinetics in two human neoplasms, one a malignant melanoma and one a fibrosarcoma, transplanted to and grown serially in the athymic nude mutant (ANM) mouse. The experimental data obtained codrealted well with a theoretical percentage labelled mitoses curve based on the assumption that the time spent by a cell in each of the phases M, G1, S and G2 is described by four independent log-normal distributions. However, no unique second wave was defined by the experimental results. This means that only the deductions made about the duration of the G2 and S phases are reliable. The median duration of tma and the fibrosarcoma, respectively. By comparing these results with results published on cell cycle studies of transplantable animal tumors and human tumors in situ, it is concluded that the cell cycle parameters of a human tumor grown in the ANM mouse are close to those of the same tumor in the donor patient.     

Generation of cells with phenotypes of both intercalated duct-type and myoepithelial cells in human parotid gland adenocarcinoma clonal cells grown in athymic nude mice

A neoplastic epithelial cell line was established from nude mice tumors grown after transplantation of surgical specimens from a human parotid gland adenocarcinoma. This cell line, which had ultrastructure similarities to salivary intercalated duct cells, was found by immunohistochemical techniques to contain amylase, but myosin was not detected. Ultrastructurally, cells of an intermediate type between intercalated ductal and myoepithelial cells were found in the transplanted tumors. Moreover, the expression of myosin in addition to the presence of amylase was detected in the tumors. These findings indicate that some transplanted tumor cells appear to be differentiating towards myoepithelial cells.     

体外诱导人羊膜上皮细胞分化为胰岛素分泌细胞的研究

目的:通过体外诱导分化实验,探讨人羊膜上皮细胞(hAECs)向胰岛素分泌细胞(ISCs)分化的能力。方法:采用胰蛋白酶消化法从人羊膜组织分离提取hAECs,用流式细胞仪和免疫细胞化学法进行鉴定。取第3代hAECs在含尼克酰胺和N2补充物的无血清培养基中诱导培养,分别于诱导不同时间采用免疫细胞化学法检测胰岛素和β2微球蛋白的表达,采用放射免疫法检测上清液中胰岛素含量,采用RT-PCR检测胰岛素mRNA和胰十二指肠同源异型盒因子-1(PDX-1)mRNA的表达。结果:①hAECs高表达CD29、CD73、CD166和CK19;②hAECs诱导组第7、142、1天胰岛素阳性细胞百分率分别为74.00%±1.73%、75.33%±1.15%和75.67%±0.58%,而对照组未见胰岛素阳性细胞;③hAECs诱导组第7、14、21天培养物上清液中胰岛素含量分别达(328.47±3.22)μIU/ml、(332.26±1.22)μIU/ml和(329.68±2.57)μIU/ml,均显著高于对照组(P均<0.01);④hAECs诱导前后均有PDX-1 mRNA和β2微球蛋白表达,胰岛素mRNA表达仅见于诱导组。结论:hAECs能分化为ISCs,在Ⅰ型糖尿病细胞移植治疗方面具有潜在应用前景。     

Vascularization of normal human thyroid tissue transplanted to nude mice

The vascularization of normal human thyroid tissue transplanted to nude, athymic mice was examined by light, electron microscopy and autoradiography after continuous infusion of 3H-thymidine during 2, 4 and 6 days after transplantation. Labelled vascular sprouts were found in the surrounding host connective tissue after 2 days, in between peripheral follicles after 4 days and in central parts of the transplants after 6 days. The autoradiographic observations indicate that the sprouts originated from the surrounding host tissue. The amount of sprouts increased up to a maximum after 2 weeks of transplantation. At this time large interfollicular areas were occupied by sprouts. At later observations (3-5 weeks) sprouts occurred together with typical fenestrated capillaries. After 7 weeks all sprouts had differentiated into mature vessels. Our observations suggest that the transplanted thyroid tissue induces the formation of vascular sprouts in the surrounding host connective tissue. The sprouts then penetrate and vascularize the thyroid tissue.     

Differentiation of human testicular embryonal carcinoma and teratocarcinoma grown in nude mice and soft-agar cultures

The differentiation pattern of two human germ cell tumors, grown in nude mice and in vitro is described. Tumor A was an embryonal carcinoma (EC) of borderline histology with characteristics of yolk sac tumor and of seminoma; tumor B was a teratocarcinoma with yolk sac elements and syncytiotrophoblastic giant cells. The morphology of an EC as well as cytogenetic characteristics were maintained during 20 passages in nude mice from tumor A and over 11 passages from tumor B. Tumor A did not grow in vitro. Cell suspensions prepared from xenografted tumor B grew into cystic embryoid bodies in semi-solid tissue culture medium. These embryoid bodies showed cuboidal and flattened cells with microvilli, junctional complexes, peripheral microfilaments, and annulated lamellae, reminiscent of the 'inner cell mass' of a blastula and of endoderm, respectively. When such colonies were transplanted into nude mice, however, only tumors with the morphology found in the transplants appeared.