A serum-free defined medium capable of supporting growth of four established human prostatic carcinoma cell lines

This paper describes a serum-free defined medium (Gc) that was initially designed to support growth of the human prostatic carcinoma cell line LNCaP. Our studies indicate that this medium formulation is capable of supporting short-term, long-term, and clonal growth of the LNCaP cell line. Component deletion experiments have shown that the three most critical components for LNCaP short-term growth are insulin, triiodothyronine (T3), and fetuin. Additionally, this medium was found to support short-term and clonal growth of three other human prostatic carcinoma cell lines, DU 145, PC-3, and ALVA-31. The availability of such a medium should aid in the distinction of the regulatory factors involved in growth and differentiation of malignant prostatic epithelium. © 1994 Wiley-Liss, Inc.     

28K antigen in the urothelium.

We have been investigating the nature of the mucosal lining of human urinary bladder. Surface material was obtained from bladders at autopsy and used for biochemical analysis. Western immunoblotting and tissue immunochemistry identified a molecule of Mr 28,000 (28K) which is present within surface cells of the urothelium (umbrella cells). Antiserum to 28K stained sections of the bladder of two patients with squamous cell carcinoma of the bladder and other patients with squamous metaplasia. We consider 28K to be a component of the normal human bladder surface that is increased in patients with squamous metaplasia and may also be present in squamous cell carcinoma of the bladder.     

Immortalization of human prostate epithelial cells by HPV 16 E6/E7 open reading frames.

BACKGROUND: The exact pathogenesis for prostate cancer is not known. Progress made in prostate cancer research has been slow, largely due to the lack of suitable in vitro models. Here, we report our work on the immortalization of a human prostate epithelial cell line and show that it can be used as a model to study prostate tumorigenesis. METHODS: Replication-defective retrovirus harboring the human papillomavirus (HPV) type 16 E6 and E7 open reading frames was used to infect primary human prostate epithelial cells. Polymerase chain reaction, followed by Southern hybridization for the HPV 16 E6/E7, Western blot for prostatic acid phosphatase, telomeric repeat amplification protocol assay for telomerase activity, two-dimensional gels for cytokeratins, and cytogenetic analysis were undertaken to characterized the infected cells. RESULTS: The retrovirus-infected cell line, HPr-1, continued to grow in culture for more than 80 successive passages. Normal primary cells failed to proliferate after passage 6. HPr-1 cells bore close resemblance to normal primary prostate epithelial cells, both morphologically and biochemically. However, they possessed telomerase activity and proliferated indefinitely. Cytogenetic analysis of HPr-1 cells revealed a human male karyotype with clonal abnormalities and the appearance of multiple double minutes. CONCLUSIONS: The HPr-1 cells expressed prostatic acid phosphatase and cytokeratins K8 and K18, proving that they were prostate epithelial cells. They were benign in nude mice tumor formation and soft agar colony formation assay. The HPr-1 cell line is an in vitro representation of early prostate neoplastic progression.     

Characterization of an immortalized human vaginal epithelial cell line

PURPOSE: Adherence of type 1 piliated Escherichia coli to vaginal mucosa plays a major role in the pathogenesis of ascending urinary tract infections (UTIs) in women. Progress in understanding the mechanism of adherence to the vaginal surface could be enhanced by the utilization of well-characterized vaginal epithelial cells. The objective of this study was to immortalize vaginal epithelial cells and study their bacterial adherence properties. MATERIALS AND METHODS: Primary vaginal cells were obtained from a normal post-menopausal woman, immortalized by infection with E6/E7 genes from human papillomavirus 16 (HPV 16) and cultured in serum free keratinocyte growth factor medium. RESULTS: Positive immunostaining with a pool of antibodies to cytokeratins 1, 5, 10 and 14 (K1, K5, K10 and K14) and to K13 confirmed the epithelial origin of these cells. The immortalized cells showed binding of type 1 piliated E. coli in a pili specific and mannose sensitive manner. CONCLUSION: This model system should facilitate studies on the interaction of pathogens with vaginal mucosal cells, an essential step in the progression of ascending UTIs in women.     

Primary culture of human bladder carcinomas and establishment of human bladder carcinoma cell line by serum-free culture

It was possible to cultivate cells from bladder carcinoma tissues in 4 cases out of 6 without the overgrowth of fibroblast. A new human transitional cell carcinoma cell line (HAMT-1) was established in longterm tissue culture by using serum-free medium (BEM-841) which had been developed by us. The tissue for culture was taken from a 61-year-old Japanese male with grade 3 transitional cell carcinoma of the bladder. The microscopic features of the cell in cultures and of the tumors developed in nude mice resembled closely that of the original tumor. Electron microscopy of the cultured cells and the tumors developed in nude mice revealed characteristics of the epithelial origin of these cells with microvilli, junctional complex and scarce filament formation. Blood TPA level of the nude mouse with the transplanted tumor was equally high as that of the patient from whom the original tumor had been taken. The cells were anchorage independent in the serum-free medium but anchorage dependent in the medium containing 5% FCS. Anchorage dependency could not be restored by the addition of collagen and fibronectin. The doubling time of the cells were 18-20 hours. The chromosome counts of the cell line ranged from 59-78 with a modal count of 74.     

Human papillomavirus DNA sequences in cell lines derived from head and neck squamous cell carcinomas.

There is increasing evidence that human papillomaviruses (HPV) have a casual role in some neoplasms in human beings. As examples, DNA of HPV types 16, 18, and 31 are frequently present in genital cancers in humans. Recently, oncogenic HPV types have also been identified in neoplasms of the head and neck, including verrucous carcinoma of the larynx, squamous cell carcinomas of the oral cavity and larynx, and inverted papillomas of the nose. These findings and our resource of an extensive panel of head and neck squamous cell carcinoma (HNSCC) cell lines led us to begin to investigate how frequently HPV DNA was present in these tumor cell lines. For initial analysis, twenty-two HNSCC cell lines derived from 20 patients' tumors were selected as representative of our tumor cell line panel with respect to diversity of primary site, tumor stage, patient age, sex, and clinical course. For Southern analysis, cell line DNA was tested for hybridization with DNA probes for HPV types 6, 11, 16, 18, and 31. Polymerase chain reaction (PCR) analysis was also performed on five tumor cell lines using types 6, 11, 16, 18, and 52 as probes. Southern blot analysis revealed HPV-specific signals in two of the 22 HNSCC cell lines tested. One of these, UM-SCC-23, was HPV 31 positive, which to our knowledge is the first identification of HPV 31 in HNSCC. UM-SCC-63, the other HPV-positive tumor identified by Southern analysis, hybridized with both type 18 and 31. Of the five tumor cell lines tested with PCR, two were HPV positive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential cyclic AMP responses to calcitonin among human ovarian carcinoma cell lines: a calcitonin-responsive line derived from a rare tumor type

Several human tumor cell lines have been reported to have specific receptors for calcitonin (CT) and CT-responsive adenylate cyclase. In order to correlate patterns of responsiveness to CT, parathyroid hormone (PTH) and prostaglandin E2 (PGE2) with tumor morphology and intermediate filament protein expression, we examined four human ovarian tumor cell lines (BIN-16, BIN-22, BIN-53, BIN-67) which had been cultured from cells of metastatic foci. In two cell lines (BIN-53 and -16) there were small increases in cAMP content after exposure to CT and in three cell lines (BIN-53, -16, and -22) larger increases with PGE2. There was no cAMP response in any of the cells to PTH. In BIN-67 cells, however, CT induced a striking (greater than 20-fold) increase in cAMP content. Histologically, the CT-nonresponsive tumor lines were derived from serous adenocarcinomas while the CT-responsive tumor line was from a rare small cell carcinoma. Gel electrophoretic and immunofluorescence microscopic analyses had previously disclosed that the CT-nonresponsive cell lines contained high levels of simple epithelial keratins and no or very low levels of vimentin (characteristic of ovarian surface epithelial cells), while the CT-responsive cell line contained almost exclusively vimentin. Thus, cells cultured from a rare type of ovarian tumor were CT-responsive and were distinguishable from CT-nonresponsive ovarian tumor cells by initial tumor histology and intermediate filament protein expression.     

Characterization of a mouse cortical collecting duct cell line.

A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepithelial resistance (885.7 +/- 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 +/- 3.5 mV. The associated short-circuit current (SCC) averaged 71.8 +/- 10.3 microA/cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 +/- 0.9 and B = 127.4 +/- 0.4; [K+], L = 8.6 +/- 0.6 and B = 2.1 +/- 0.3; [Cl], L = 68.6 +/- 5.8 and B = 101.8 +/- 6.6; [HCO3], L = 15.5 +/- 1.5 and B = 8.6 +/- 1.2; while pH was 7.16 +/- 0.03 at the luminal and 6.94 +/- 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+.(ABSTRACT TRUNCATED AT 250 WORDS)     

G250启动子的克隆及其在人肾癌细胞中的特异生物活性

目的 克隆G250启动子并插入pG3-Basic真核表达载体,探讨G250启动子在肾癌细胞中的特异生物活性.方法 提取宫颈癌Hela细胞总DNA作为模板,设计两对引物进行PCR,获取两段不同长度的G250启动子片段.测序后分别将获取的551 bp和218 bp启动子片段克隆到pGL3-Basic载体中,构建双荧光素酶检测质粒pGLB-G551和pGLB-G218.将pGL3-Basic、pGLB-G551、pGLB-G218各1μg转染G250阳性宫颈癌Hela细胞、肾癌Ketr-3细胞.运用双荧光素酶检测技术,确定G250启动子的转录活性.结果 电泳与测序结果 显示,两段G250启动子片段与报道序列一致.酶切与测序结果 显示,质粒pGLB-G551、pGLB-G218构建成功.转染pGLB-G551、pGLB-G218的Hela细胞相对荧光素酶活性分别为pGL3-Basic的11.07倍、10.36倍.Ketr-3细胞分别为pGL3-Basic的5.17倍、4.13倍.与空载体pGL3-Basic比较,pGLB-G551和pGLB-G218的相对荧光素酶活性均明显增强(P<0.05).pGLB-G551和pGLB-G218比较,相对荧光素酶活性差异无统计学意义(P>0.05).结论 成功克隆出肾癌特异性G250启动子,并证明具有良好转录活性,218bp的启动子片段已具有G250启动子的所有启动效果.     

K+ channel inhibition accelerates intestinal epithelial cell wound healing

Restitution is the process by which superficial interruptions in the gastrointestinal mucosa are repaired by the flattening and spreading of epithelial cells surrounding the damage. During this process, mucosal epithelial cells undergo extensive reshaping and cytoskeletal remodeling. K(+) channels, located primarily on the basolateral surface of gut epithelial cells, are central to both actin polymerization, via their control of membrane potential, and cell volume regulation. We questioned whether K(+) channels are involved in restitution using an in vitro model of intestinal epithelium, monolayers of the human colon carcinoma cell line T84. We report that pharmacologic K(+) channel inhibition accelerates wound healing in T84 cell monolayers. Both Ca(++)-dependent and constitutively active channels are involved, as indicated by the sensitivity to clotrimazole, charybdotoxin, and barium. The ability of clotrimazole to accelerate wound resealing was also observed in Caco-2 cell sheets. Pharmacologic stimulation of K(+) channel activity had no effect on the repair rate. Analysis of the resealing process by time lapse and confocal microscopy revealed that K(+) channel inhibitors abolished the initial wound retraction, briefly accelerated the repair rate, and altered the shape of the cell sheet abutting the injury during the early phase of resealing. We hypothesize that K(+) channel inactivation interrupts the coregulation of f-actin polymerization and volume control that is initiated by the healing process.     

Fibrinolytic activity of in vitro cultivated human bladder cell lines

Summary Three human bladder carcinoma cell lines, T 24, RT 4, and MANO, a human bladder nonmalignant epithelial cell line, HCV-29, and a human lung fibroblast line, 460 Hl, were investigated for their ability to induce fibrinolytic, urokinase and plasmin inhibitory activities in cell culture, using serum-free medium, for up to 36 h. Generally, the non-malignant cell line and the fibroblast line had a greater ability to produce urokinase inhibitor than did the malignant cell lines. The amount produced varied greatly between cells and over the study period. A low concentration of plasminogen activator, immunologically identical with urokinase, and its accumulation in culture supernate were found with RT 4 after 12 h and 24 h cultivations, whereas no plasminogen activator was detected in all other cell lines for periods up to 36 h. No plasmin, non-specific protease or plasmin inhibitory activities were detected in any of the supernates from the cell lines.     

Expression of cell adhesion molecules in an established and characterized new human renal cell cancer line,CCF-RC7

In order to investigate the importance of cell adhesion molecules (CAMs) in renal cell carcinoma (RCC), a cell line, designated as CCF-RC7, was established from a human RCC of the clear cell type. CCF-RC7 was passaged over 50 times in vitro for 31/2 years. The cell line has an epithelial morphology and a doubling time of 30 h, forming colonies in soft agar with an average efficiency of 10.4% and producing clear cell tumors in athymic nude mice. CCF-RC7 cells have an aneuploid-hypotetraploid karyotype with a modal chromosome number of 82 and rearrangements in chromosomes 9, 12 and 14. Immunohistochemical and flow immunocytometric analyses revealed high expression of ICAM-1 (CD54), and Hermes antigen (CD44), which was significantly upregulated by cytokine and PMA treatment. VLA-4 was expressed on approximately 20% of tumor cells and could not be altered by cytokine or PMA stimulation. High expression of sialyl Lewis X was also demonstrated by immunohistological examination. This newly characterized cell line will serve as a useful model for the study of CAMs during hematogenous metastasis and host defense mechanisms in human RCC.     

Surface Papillary Epithelial Hyperplasia (Rough Mucosa) is a Helpful Clue for Identification of Polymorphous Low-Grade Adenocarcinoma

The purpose of this study is to evaluate surface papillary epithelial hyperplasia, a microscopic finding that corresponds to the clinical finding of rough or stippled mucosa, as a predictor of polymorphous low-grade adenocarcinoma (PLGA). We conducted a retrospective review of minor salivary gland neoplasms submitted to our biopsy service from 1991 to 2013. Our review was limited to lesions involving the oral cavity/soft palate with the following diagnoses: PLGA, pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC). A total of 202 minor salivary gland neoplasms were included in the study. Among cases in which surface epithelium was present for evaluation (n = 112), surface papillary epithelial hyperplasia was evident in 30 % of PLGA and 1 % of non-PLGA (i.e., MEC, ACC, PA). The greater frequency of surface papillary epithelial hyperplasia in the PLGA versus non-PLGA cases and in the benign versus malignant cases was significant (p = .0001 and p = .041, respectively). The sensitivity and specificity of papillary epithelial hyperplasia for PLGA were 30 % (95 % confidence interval (CI) 11.97–54.27 %) and 99 % (95 % CI 94–99.82 %), respectively. The clinical presentation of PLGA appeared relatively nonspecific, with all analyzed tumor types exhibiting a predilection for females, middle-aged to older adults, palatal location, pink/tan/normal color, and firm consistency. In conclusion, papillary epithelial hyperplasia was evident in only a minority of PLGA. However, when present within the context of a palatal salivary gland neoplasm, it appears to indicate a high probability of PLGA. Accordingly, rough mucosa may be a useful clinical pearl for identification of PLGA.     

UNME/K1: an IgG2a monoclonal antibody specific to cytokeratin of human urinary bladder squamous cell carcinoma

Summary The main distinctive feature of carcinoma in schistosomal bladder is keratinized squamous cell carcinoma. Keratins/cytokeratins constitute a multigeneic family of structurally related polypeptide markers for the malignant state of epithelial cells. A monoclonal antibody (UNME/K1) regognizing keratins associated with squamous cell carcinoma of the human urinary bladder was generated at the Urology and Nephrology Center, Mansoura, Egypt (UNME), by fusion of spleenocytes from a BALB/c mouse immunized with a keratin extract (K1) of human squamous cell carcinoma and P3X63Ag8/U1 syngeneic myeloma cells. UNME/K1 was purified by a protein-A affinity column and was of the IgG2a type, as determined by immunoelectrophoresis and gel diffusion techniques. When tested against keratins of different types of urinary bladder tumors using enzym linked immunosorbent assay (ELISA), UNME/K1 reacted only with the high molecular weight keratin of squamous cell carcinoma and showed selectivity towards specific histopathological grades of tumors.     

维生素E琥珀酸酯对人肝癌细胞凋亡和侵袭性

目的研究维生素E琥珀酸酯（VES）诱导体外培养人肝癌细胞凋亡以及对肝癌细胞侵袭性的抑制作用。方法自2004年至2005年7月,体外培养SMMC7721人肝癌细胞系,在培养液中加入VES共育,用等量的生理盐水作为对照组。培养6、24、48h后采用流式细胞仪检测肝癌细胞的凋亡,小孔趋化试验检测肝癌细胞的体外侵袭性变化。结果在加入VES后肝癌细胞的凋亡率与对照组相比明显增加。在48h加入VES的实验组与对照组相比,肝癌细胞的小孔趋化试验明显受到抑制。结论VES具有体外诱导人肝癌细胞系凋亡以及抑制肝癌细胞体外侵袭性的作用,是潜在的抗肿瘤因子。     

Epidermal Growth Factor Receptor Expression in Spindle Cell Carcinomas of the Head and Neck

Spindle cell carcinoma (SpCC) is an uncommon head and neck squamous cell carcinoma (SCC) variant consisting of spindled and/or pleomorphic cells with epithelial differentiation. Epidermal growth factor receptor (EGFR) is expressed by >90 % of conventional SCC, and high level expression is associated with a poorer prognosis. Anti-EGFR therapies are commonly used to treat head and neck SCC. However, no studies have evaluated EGFR expression in SpCC. Cases of SpCC were retrieved from department files. The diagnosis required either a biphasic lesion with a squamous neoplastic component, or a purely spindle cell or pleomorphic tumor with immunohistochemical positivity for epithelial markers. EGFR immunohistochemistry was performed and was quantified in quartiles. Medical records were reviewed for clinical follow up information. EGFR was expressed in 21/30 (70 %) cases, including in the squamous component in 18/19 (95 %) and the spindle cell component in only 12/30 (40 %). Where the spindle cell component was positive, the intensity and distribution were lower than for the squamous component. Recurrent tumors were predominantly (80–90 %) of the spindle cell component, and had low (or absent) EGFR expression. Kaplan–Meier survival analysis showed no statistically significant differences in overall or disease free survival between the EGFR expressing and non-expressing groups (p = 0.414 and 0.19, respectively). SpCCs of the head and neck have a poor prognosis, and markedly reduced EGFR expression. EGFR-specific therapies may not be ideal for SpCC patients, which may lack EGFR expression, but further studies are needed.

Electronic supplementary material

The online version of this article (doi:10.1007/s12105-014-0604-y) contains supplementary material, which is available to authorized users.     

METHOTREXATE RESISTANCE IN HUMAN UROEPITHELIAL CELLS WITH p53 ALTERATIONS

Purpose

Bladder cancers are frequently treated with combination chemotherapy that includes methotrexate (MTX). The development of drug resistance is a common problem in treatment of bladder cancers. We tested if the status of p53 and/or pRb affects the development of MTX resistance in bladder epithelial cell lines.

Materials and Methods

We used two isogeneic sets of cell lines in which we manipulated the status of p53 and/or pRb by transformation with Human Papillomavirus (HPV) E6 and/or E7 or with a transdominant TP53 mutant (TP53 sup 143). One series of isogeneic origin was derived from normal human uroepithelial cells (HUC), and the other was derived from a human transitional cell carcinoma (TCC). Cell lines with p53 and/or pRb alterations were cultured for six months while increasing the MTX concentration in each line, as resistance developed.

Results

Two cell lines with both pRb and p53 alterations, alpha E6/E7-HUC and alpha E7-HUC^p53mu, acquired the greatest resistance (750 nM) to MTX. One line with p53 loss, E6-TCC#10, acquired intermediate resistance (500 nM), while two lines, alpha E7-HUC and E7-TCC#10, with altered pRb but wildtype p53, showed low levels of MTX resistance (125 nM and 80 nM, respectively). Two clear mechanisms of MTX resistance were identified. All five MTX resistant cell lines showed altered uptake of MTX. In addition, two of five MTX resistant cell lines, both with altered p53, showed dihydrofolate reductase (DHFR) amplification.

Conclusions

p53 alteration increases the risk for development of drug resistance by both DHFR amplification and altered MTX transport in transformed human bladder epithelial cell lines.     

丹参对马兜铃酸致肾小管上皮细胞损害的保护作用研究

目的:探讨丹参对马兜铃酸(aristolochic acid,AA)所致肾小管上皮细胞损害的保护作用.方法:以95%DMEM培养液加5%小牛血清培养肾小管上皮细胞株(NRK-52E),加入不同浓度的丹参和马兜铃酸,采用MTT法来观察肾小管上皮细胞存活以及生长的状况;用碘化丙啶(PI)染色方法确认细胞凋亡的发生.结果:马兜铃酸浓度超过10 mg/ml时肾小管上皮细胞的活力显著抑制(P＜0.05).以丹参3.0、15、30、60 mg/ml处理后的肾小管上皮细胞再加入AA(10 mg/ml浓度),则肾小管上皮细胞的活力明显增加,且随着丹参浓度的增加其作用愈趋明显(P＜0.01),丹参明显抑制AA所造成肾小管上皮细胞的凋亡(P＜0.01).结论:丹参(3.0、15、30、60 mg/ml)通过减少马兜铃酸致肾小管上皮细胞的凋亡,可明显保护肾小管上皮细胞,且其作用随着丹参浓度的增加而加强.     

Evaluation of intercellular communication of human prostatic epithelium

Intercellular communication (IC) among epithelial and/or interstitial cells of human prostate was evaluated. IC was measured by the dye transfer method. In brief, fluorescent lucifer yellow CH was microinjected into an individual cell and communication was measured by scoring the number of surrounding fluorescent cells. Cells used here were outgrowth cells from small tips of tissues of various prostatic diseases and established prostatic carcinoma cell line PC-3. To identify the epithelial cells, keratin staining was done. In prostatitis and benign prostatic hyperplasia, epithelial cells grew in five of six cases cultured and extent of maximum IC was from 18 to 46 cells (mean = 26 cells) at 4 to 6 culture days. Three specimens of separate cases cultured for more than one month had a maximum IC of more than 100 cells. In prostatic carcinoma, epithelial cells grew in 7 of 10 cases cultured and extent of maximum IC was from 6 to 38 cells (mean = 18 cells) at 3 to 6 days. The extent of IC among prostatic carcinoma cells was lower than that of benign prostatic diseases, though not significantly, this indicates the possibility that benign cells were present among the malignant cells in the specimens examined above. Prostatic carcinoma cells cultured from metastatic lesions of bone (PC-3) and lymph nodes had maximum IC of 3 to 5 cells. IC was very limited or absent between benign prostatic epithelium and PC-3 cells. No IC was present between prostatic epithelial and interstitial cells. Interstitial cells prepared from prostatic benign and malignant diseases similarly revealed a high extent of IC.(ABSTRACT TRUNCATED AT 250 WORDS)