Gene expression profile analysis in laryngeal cancer by high-density oligonucleotide microarrays.

The assessment of gene expression profile in laryngeal cancer shall allow to implement molecular biology methods in diagnostics, as well as in prognosis of the course of disease. Thus, it may influence the choice of the most optimal decisions in regards to the method of treatment, extent of surgical procedure, or the necessity of adding post-operative radiotherapy. The aim of the project was to analyse the gene expression profile of laryngeal cancer using oligonucleotide microarrays, aiming to derive novel molecular markers for that carcinoma. The study comprised a group of 14 patients (12 males and 2 females) with squamous cell laryngeal carcinoma, diagnosed and surgically treated between 2005 - 2007 in the ENT Department of the Silesian Medical University in Katowice, Poland. RNA was isolated from frozen tissue fragments. To assess gene expression profile, high density oligonucleotide microarrays (Affymetrix U 133 Plus 2.0) were applied, with over 54 thousand probesets for over 47 thousand transcripts. Four genes, previously not assesed in diagnostic context in laryngeal carcinoma, seemed to be valuable markers of that neoplasm. These are: metalloproteinase ADAM12, cycline-dependent kinase 2 - CDK2, kinesine 14 - KIF14, suppressor 1 of checkpoint - CHES1.

Inhibition of porcine endogenous retroviruses (PERVs) in primary porcine cells by RNA interference using lentiviral vectors.

A potential risk in pig-to-human xenotransplantation is the transmission of PERVs to human recipients. Here we show for the first time the inhibition of PERV expression in primary porcine cells by RNA interference using lentiviral vectors. Cells were transduced with lentiviral vectors coding for short hairpin (sh) RNAs directed against PERV. In all primary porcine cells studied and in the porcine kidney cell line PK-15, expression of PERV-mRNA was significantly reduced as measured by real-time PCR. Most importantly, expression of PERV proteins was almost completely suppressed, as shown by Western blot analysis. Thus, lentiviral shRNA vectors could be used to knockdown PERV expression and create transgenic pigs with a reduced risk of PERV transmission during xenotransplantation.

Reconstitution of telomerase activity combined with HPV-E7 expression allow human preadipocytes to preserve their differentiation capacity after immortalization.

Overexpression of SV40 T-antigen (SV40 T-Ag) has been widely used to overcome replicative senescence of human primary cells and to promote cell immortalization. However, in the case of certain cell types, such as preadipocytes, the differentiation process of immortalized cells is blocked by SV40 T-Ag expression. In this study, human telomerase reverse transcriptase (hTERT) and papillomavirus E7 oncoprotein (HPV-E7) genes were coexpressed in human preadipocytes to test whether this combination could maintain cell differentiation capacity after immortalization. We demonstrated that the HPV-E7/hTERT expressing preadipocytes displayed an indefinite life span. Interestingly, immortalized cells were diploid and presented no chromosomic alterations. These immortalized cells were able to accumulate and hydrolyze intracellular triglycerides and to express adipocyte markers. These data demonstrate, for the first time, that coexpression of hTERT and HPV-E7 in human preadipocytes allows cells not only to display an indefinite life span but also to retain their capacity to differentiate.

[Findings in a high-risk group following asbestos exposure. Benefit of ENT examinations in patients with long-term exposure to asbestos]. / Befunde in einem Hochrisikokollektiv nach Asbestexposition Nutzen von HNO-ärztlichen Untersuchungen ehemals asbeststaubexponierter Personen.

BACKGROUND AND OBJECTIVE: Laryngeal diseases caused by exposure to asbestos are listed in the current German list of occupational diseases under number 4104. Parallel to a multicenter study to evaluate whether a CT scan should be included in the examinations for occupational diseases according to the German surveillance guidelines, an additional ENT examination was evaluated. PATIENTS AND METHODS: One hundred workers with a mean exposure time to asbestos of 20.9 years were given a complete ENT examination in 4 consecutive years (1993-1996). Radiological signs for asbestosis were observed in 21 cases and 58 participants had pleural affections caused by asbestos. Significant nicotine abuse was reported by 15 persons: 61 participants were ex-smokers and 24 had never smoked. Regular alcohol consumption was reported by 90% (11% more than 80 g/day). RESULTS: As documented in the literature, we found a high prevalence of laryngitis, especially in smoking patients. One patient had early laryngeal cancer (T1). CONCLUSION: The integration of an ENT examination into the German surveillance guidelines for occupational diseases should be discussed for patients with a high exposure to asbestos.

Matrix metalloproteinase/integrin interactions as target for anti-angiogenic treatment strategies.

Modulation of the balance between pro- and antiangiogenic factors holds great promise for the treatment of a broad spectrum of human disease ranging from ischemic heart disease to cancer. This requires both the identification of angiogenic regulators and their efficient delivery to target organs. Here, we demonstrate the use of a noncatalytic fragment of matrix metalloproteinase 2 (termed PEX) delivered by lentiviral vectors in different angiogenesis models. Transduction of human endothelial cells with PEX virus suppressed endothelial invasion and formation of capillary-like structures without affecting chemotaxis in vitro. Lentiviral delivery of PEX blocked basic fibroblast growth factor-induced matrix metalloproteinase 2 activation and angiogenesis on chicken chorioallantoic membranes. PEX expression also inhibited tumor-induced angiogenesis and tumor growth in a nude mouse model. Thus, our study shows that lentiviral vectors can deliver sufficient quantities of antiangiogenic substances to achieve therapeutic effects in vivo.

Gene therapy: promises and problems.

Gene therapy can be broadly defined as the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient. One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells. Based on the nature of the viral genome, these gene therapy vectors can be divided into RNA and DNA viral vectors. The majority of RNA virus-based vectors have been derived from simple retroviruses like murine leukemia virus. A major shortcoming of these vectors is that they are not able to transduce nondividing cells. This problem may be overcome by the use of novel retroviral vectors derived from lentiviruses, such as human immunodeficiency virus (HIV). The most commonly used DNA virus vectors are based on adenoviruses and adeno-associated viruses. Although the available vector systems are able to deliver genes in vivo into cells, the ideal delivery vehicle has not been found. Thus, the present viral vectors should be used only with great caution in human beings and further progress in vector development is necessary.

Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo.

The Cre recombinase (Cre) from bacteriophage P1 is an important tool for genetic engineering in mammalian cells. We constructed lentiviral vectors that efficiently deliver Cre in vitro and in vivo. Surprisingly, we found a significant reduction in proliferation and an accumulation in the G(2)/M phase of Cre-expressing cells. To minimize the toxic effect of Cre, we designed a lentiviral vector that integrates into the host genome, expresses Cre in the target cell, and is subsequently deleted from the genome in a Cre-dependent manner. Thus, the activity of Cre terminates its own expression (self-deleting). We showed efficient modification of target genes in vitro and in the brain after transduction with the self-deleting vectors. In contrast to sustained Cre expression, transient expression of Cre from the self-deleting vector induced significantly less cytotoxicity. Such a self-deleting Cre vector is a promising tool for the induction of conditional gene modifications with minimal Cre toxicity in vivo.

Cytochrome P450 expression and related metabolism in human buccal mucosa.

Constituents in food and fluids, tobacco chemicals and many drugs are candidates for oral absorption and oxidative metabolism. On this basis, the expression of cytochrome P450 isozymes (CYPs) and the conversion of CYP substrates were analysed in reference to buccal mucosa. A RT-PCR based analysis of human buccal tissue from 13 individuals demonstrated consistent expression of mRNA for the CYPs 1A1, 1A2, 2C, 2E1, 3A4/7 and 3A5. CYP 2D6 was expressed in six out of the 13 specimens, whereas all samples were negative for 2A6 and 2B6. Serum-free monolayer cultures of the Siman virus 40 large T-antigen-immortalized SVpgC2a and the carcinoma SqCC/Y1 buccal keratinocyte lines expressed the same CYPs as tissue except 3A4/7 and 3A5 (SVpgC2a), and 2C, 2D6 and 3A4/7 (SqCC/Y1). Dealkylation of ethoxyresorufin and methoxyresorufin in both normal and transformed cells indicated functional 1A1 and 1A2, respectively. SVpgC2a showed similar activity as normal keratinocytes for both substrates, whereas SqCC/Y1 showed about 2-fold lower 7-ethoxyresorufin O-deethylation and 7-methoxyresorufin O-demethylation activities. SVpgC2a showed detectable and many-fold higher activity than the other cell types towards chlorzoxazone, a substrate for 2E1. Absent or minute catalytic activity of 2C9, 2D6 and 3A4 in the various cell types was indicated by lack of detectable diclofenac, dextromethorphan and testosterone metabolism (<0.2-0.5 pmol/min/mg). Metabolic activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the mycotoxin aflatoxin B1 (AFB1) to covalently bound adducts was indicated by autoradiographic analysis of both monolayer and organotypic cultures of SVpgC2a. In contrast, SqCC/Y1 showed lower or absent metabolic activity for these substrates. Finally, measurements of various non-reactive AFB1 metabolites indicated rates of formation <0.1 pmol/min/mg in both normal and transformed cells. The results indicate presence of several CYPs of which some may contribute to significant xenobiotic metabolism in human buccal epithelium. Notably, metabolic activation of AFB1 was not previously implicated for oral mucosa. Further, the results show that CYP-dependent metabolism can be preserved or even activated in immortalized keratinocytes. Metabolic activity in SVpgC2a under both monolayer and organotypic culture conditions suggests that this cell line may be useful to pharmaco-toxicological and carcinogenesis studies.

Mechanisms of NO/cGMP-dependent vasorelaxation.

Both cGMP-dependent and -independent mechanisms have been implicated in the regulation of vascular tone by NO. We analyzed acetylcholine (ACh)- and NO-induced relaxation in pressurized small arteries and aortic rings from wild-type (wt) and cGMP kinase I-deficient (cGKI(-/-)) mice. Low concentrations of NO and ACh decreased the spontaneous myogenic tone in wt but not in cGKI(-/-) arteries. However, contractions of cGKI(-/-) arteries and aortic rings were reduced by high concentrations (10 micromol/L) of 2-(N:, N-diethylamino)-diazenolate-2-oxide (DEA-NO). Iberiotoxin, a specific blocker of Ca(2+)-activated K(+) (BK(Ca)) channels, only partially prevented the relaxation induced by DEA-NO or ACh in pressurized vessels and aortic rings. DEA-NO increased the activity of BK(Ca) channels only in vascular smooth muscle cells isolated from wt cGKI(+/+) mice. These results suggest that low physiological concentrations of NO decrease vascular tone through activation of cGKI, whereas high concentrations of DEA-NO relax vascular smooth muscle independent of cGKI and BK(Ca). NO-stimulated, cGKI-independent relaxation was antagonized by the inhibition of soluble guanylyl cyclase or cAMP kinase (cAK). DEA-NO increased cGMP to levels that are sufficient to activate cAK. cAMP-dependent relaxation was unperturbed in cGKI(-/-) vessels. In conclusion, low concentrations of NO relax vessels by activation of cGKI, whereas in the absence of cGKI, NO can relax small and large vessels by cGMP-dependent activation of cAK.

Suppression of angiogenesis by lentiviral delivery of PEX, a noncatalytic fragment of matrix metalloproteinase 2.

Modulation of the balance between pro- and antiangiogenic factors holds great promise for the treatment of a broad spectrum of human disease ranging from ischemic heart disease to cancer. This requires both the identification of angiogenic regulators and their efficient delivery to target organs. Here, we demonstrate the use of a noncatalytic fragment of matrix metalloproteinase 2 (termed PEX) delivered by lentiviral vectors in different angiogenesis models. Transduction of human endothelial cells with PEX virus suppressed endothelial invasion and formation of capillary-like structures without affecting chemotaxis in vitro. Lentiviral delivery of PEX blocked basic fibroblast growth factor-induced matrix metalloproteinase 2 activation and angiogenesis on chicken chorioallantoic membranes. PEX expression also inhibited tumor-induced angiogenesis and tumor growth in a nude mouse model. Thus, our study shows that lentiviral vectors can deliver sufficient quantities of antiangiogenic substances to achieve therapeutic effects in vivo.

Functional characteristics of urinary tract smooth muscles in mice lacking cGMP protein kinase type I.

Nitric oxide (NO)-mediated smooth muscle relaxation is mediated by cGMP through activation of cGMP-dependent protein kinase I (cGKI). We studied the importance of cGKI for lower urinary tract function in mice lacking the gene for cGKI (cGKI-/-) and in litter-matched wild-type mice (cGKI+/+) in vitro and in vivo. cGKI deficiency did not result in any changes in bladder gross morphology or weight. Urethral strips from cGKI-/- mice showed an impaired relaxant response to nerve-derived NO. The cGMP analog 8-bromo-cGMP (8-BrcGMP) and the NO-donor SIN-1 relaxed the wild-type urethra (50-60%) but had only marginal effects in the cGKI-deficient urethra. Bladder strips from cGKI-/- mice responded normally to electrical field stimulation and to carbachol but not to 8-BrcGMP. In vivo, the cGKI-deficient mice showed bladder hyperactivity characterized by decreased intercontraction intervals and nonvoiding bladder contractions. Loss of cGKI abolishes NO-cGMP-dependent relaxations of urethral smooth muscle and results in hyperactive voiding. These data suggest that certain voiding disturbances may be associated with impaired NO-cGKI signaling.

Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells.

Thiabendazole (TBZ), an anthelmintic and fungicide benzimidazole, was recently demonstrated to be extensively metabolized by cytochrome P450 (CYP) 1A2 in man and rabbit, yielding 5-hydroxythiabendazole (5OH-TBZ), the major metabolite furtherly conjugated, and two minor unidentified metabolites (M1 and M2). In this study, exposure of rabbit and human cells to 14C-TBZ was also shown to be associated with the appearance of radioactivity irreversibly bound to proteins. The nature of CYP isoforms involved in this covalent binding was investigated by using cultured rabbit hepatocytes treated or not with various CYP inducers (CYP1A1/2 by beta-naphthoflavone, CYP2B4 by phenobarbital, CYP3A6 by rifampicine, CYP4A by clofibrate) and human liver and bronchial CYP-expressing cells. The covalent binding to proteins was particularly increased in beta-naphthoflavone-treated rabbit cells (2- to 4-fold over control) and human cells expressing CYP1A2 (22- to 42-fold over control). Thus, CYP1A2 is a major isoenzyme involved in the formation of TBZ-derived residues bound to protein. Furthermore, according to the good correlation between covalent binding and M1 or 5OH-TBZ production, TBZ would be firstly metabolized to 5OH-TBZ and subsequently converted to a chemically reactive metabolic intermediate binding to proteins. This metabolic activation could take place preferentially in liver and lung, the main biotransformation organs, rather than in intestines where TBZ was shown to be not metabolized. Moreover, TBZ was rapidly transported by passive diffusion through the human intestinal cells by comparison with the protein-bound residues which were not able to cross the intestinal barrier. Consequently, the absence of toxicity measured in intestines could be related to the low degree of TBZ metabolism and the lack of absorption of protein adducts. Nevertheless, caution is necessary in the use of TBZ concurrently with other drugs able to regulate CYP1A2, particularly in respect to liver and lung tissues, recognised as sites of covalent-binding.

Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures.

BACKGROUND AND AIM: Intestinal epithelial cells (IEC) are thought to participate in the mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis. Reactivity of IEC to bacterial signals may depend on interactions with immunocompetent cells. To address the question of whether non-pathogenic bacteria modify the immune response of the intestinal epithelium, we co-cultivated enterocyte-like CaCO-2 cells with human blood leucocytes in separate compartments of transwell cultures. METHODS: CaCO-2/PBMC co-cultures were stimulated with non-pathogenic bacteria and enteropathogenic Escherichia coli. Expression of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, IL-8, monocyte chemoattracting protein 1 (MCP-1), and IL-10 was studied by enzyme linked immunosorbent assays (cytokine secretion) and by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: Challenge of CaCO-2 cells with non-pathogenic E coli and Lactobacillus sakei induced expression of IL-8, MCP-1, IL-1beta, and TNF-alpha mRNA in the presence of underlying leucocytes. Leucocyte sensitised CaCO-2 cells produced TNF-alpha and IL-1beta whereas IL-10 was exclusively secreted by human peripheral blood mononuclear cells. CaCO-2 cells alone remained hyporesponsive to the bacterial challenge. Lactobacillus johnsonii, an intestinal isolate, showed reduced potential to induce proinflammatory cytokines but increased transforming growth factor beta mRNA in leucocyte sensitised CaCO-2 cells. TNF-alpha was identified as one of the early mediators involved in cellular cross talk. In the presence of leucocytes, discriminative activation of CaCO-2 cells was observed between enteropathogenic E coli and non-pathogenic bacteria. CONCLUSION: The differential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes. These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of cellular interactions.

Erectile dysfunction in cyclic GMP-dependent kinase I-deficient mice.

The generation of nitric oxide (NO) in penile erectile tissue and the subsequent elevation of cyclic GMP (cGMP) levels are important for normal penile erection. Current treatments of erectile dysfunction elevate either cGMP levels by blocking cGMP degrading phosphodiesterase 5 or cyclic AMP (cAMP) levels by intrapenile injection of prostaglandin E1. The molecular target or targets of cGMP in erectile tissue and the role of cAMP for normal penile erection are not known. Herein, we report that mice lacking cGMP-dependent kinase I (cGKI) have a very low ability to reproduce and that their corpora cavernosa fail to relax on activation of the NO/cGMP signaling cascade. Elevation of cAMP by forskolin, however, induces similar relaxation in normal and cGKI-null corpus cavernosum. In addition, sperm derived from cGKI-null mice is normal, can undergo acrosomal reactions, and can efficiently fertilize eggs. Altogether, these data identify cGKI as the downstream target of cGMP in erectile tissue and provide evidence that cAMP signaling cannot compensate for the absence of the cGMP/cGKI signaling cascade in vivo.

Impaired relaxation of stomach smooth muscle in mice lacking cyclic GMP-dependent protein kinase I.

1. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP- and vasoactive intestinal polypeptide (VIP)/adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-signalling pathways in the gastric fundus of wild type and cGKI-deficient mice. 2. Using immunohistochemistry, similar staining patterns for NO-synthase, cyclic GMP- and VIP-immunoreactivities were found in wild type and cGKI-deficient mice. 3. In isolated, endothelin-1 (3 nM - 3 microM)-contracted, muscle strips from wild type mice, electrical field stimulation (1 - 16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI-deficient mice only the slowly developing relaxation was observed. 4. The responses to the NO donor, SIN-1 (10 nM - 100 microM), and to 8-Br-cyclic GMP (10 nM - 100 microM) were markedly impaired in strips from cGKI-deficient mice, whereas the responses to VIP (0.1 nM - 1 microM) and forskolin (0.1 nM - 1 microM) were similar to those in wild type mice. 5. These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP-pathway can exert their effects independently of cGKI.

Differential role of cyclic GMP-dependent protein kinase II in ion transport in murine small intestine and colon.

BACKGROUND & AIMS: The aim of this study was to determine the role of guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase (cGK) type II in intestinal fluid homeostasis under basal conditions and following exposure to cGMP-linked secretagogues, e.g., Escherichia coli heat-stable enterotoxin (STa) and guanylin. METHODS: Fluid and ion transport was determined in different segments of the intestine of wild-type and cGK II-deficient mice by ligated loop assays in vivo, and by short-circuit current and isotope flux measurements in vitro. RESULTS: Small intestinal fluid absorption in vivo was enhanced in cGK II-deficient mice under basal conditions and in the presence of STa. Furthermore, STa, guanylin, and 8-pCPT-cGMP stimulation of electrogenic anion secretion and inhibition of Na(+) absorption in vitro were markedly reduced in the small intestine from cGK II -/- mice but not in proximal colon. The type III phosphodiesterase inhibitor amrinone mimicked STa action in cGK II -/- mice, and also stimulated ion secretion in humans. CONCLUSIONS: This study shows that the cGMP/cGK II pathway regulates fluid homeostasis in the small intestine under basal conditions and mediates STa effects by both increasing anion secretion and inhibiting Na(+) absorption. It also demonstrates the presence of a cGK II-independent pathway for STa/cGMP-provoked secretion predominantly in the colon, which possibly involves a cGMP-inhibitable phosphodiesterase and/or activation of the cAMP-dependent protein kinase pathway.

Hepatic metabolism of diclofenac: role of human CYP in the minor oxidative pathways.

The aim of this study was to re-examine the human hepatic metabolism of diclofenac, with special focus on the generation of minor hydroxylated metabolites implicated in the idiosyncratic hepatotoxicity of the drug. Different experimental approaches were used: human hepatocytes, human microsomes, and engineered cells expressing single human CYP (cytochromes P450). Human hepatocytes formed 3'-hydroxy-, 4'-hydroxy-, 5-hydroxy- 4',5-dihydroxy-, and N,5-dihydroxydiclofenac, as well as several lactams. Formation of 4'- and 5-hydroxydiclofenac by human liver microsomes followed a Michaelis-Menten kinetics (Km 9 +/- 1 microM; Vmax 432 +/- 15 pmol/min/mg and Km 43 +/- 5 microM; and Vmax 15.4 +/- 0.6 pmol/min/mg, respectively). Secondary metabolites were detected after incubation of 5-hydroxydiclofenac with human liver microsomes, yielding 4',5-dihydroxydiclofenac (Km 15 +/- 1 microM; Vmax 96 +/- 3 pmol/min/mg) and small amounts of N,5-dihydroxydiclofenac (non-Michaelis-Menten kinetics). Based on microsome studies and the incubations with human hepatocytes and engineered cells, we estimated that in vivo CYP2C9 would be exclusively responsible for the 4' hydroxylation of diclofenac (>99.5%) as well as 5-hydroxydiclofenac (>97%). CYP2C9 was exclusively responsible for the formation of 3'-hydroxydiclofenac. Multiple regression analysis evidenced that the rate of production of 5-hydroxydiclofenac in human microsomes followed the algorithm: 0.040 x S-mephenytoin 4'-hydroxylation + 0.083 x tolbutamide methylhydroxylation, (multiple correlation coefficient = 0.969). However, the incubation of diclofenac with cell lines expressing different human CYP suggested that 7 isoforms could be involved. Comparison of data obtained with CYP-expressing cells and human hepatocytes suggests that CYP2C8 > CYP2C19 approximately CYP2C18 >> CYP2B6 are the isoforms implicated in the 5-hydroxylation of diclofenac in vivo.

Rearrangements of chromosome band 1p36 in non-Hodgkin's lymphoma.

We studied 850 consecutive cases of histologically ascertained pretreatment non-Hodgkin's lymphoma with cytogenetically abnormal clones. The diagnostic karyotypes revealed that 12% of these cases exhibited structural rearrangements involving chromosome band 1p36. Here, we describe the karyotypes of 53 cases containing a 1p36 rearrangement [often involving translocations of unknown material and presented as add(1)(p36)]. We used fluorescence in situ hybridization to determine the origin of the translocation partners. We report three different recurrent translocations involving 1p36. These include der(1)t(1;1)(p36;q21) (three cases), der(1)t(1;1)(p36;q25) (three cases), and der(1)t(1;9)(p36;q13) (four cases). Using cytogenetic and fluorescence in situ hybridization analyses, we have resolved the translocation partners in 31 cases. Rearrangements of band 1p36 were found among different histopathological subtypes. Alterations of 1p36 never occurred as a sole abnormality, and in 42 of 53 cases, alterations of the band 14q32 were observed. The t(14;18)(q32;q21) translocation was present in 35 cases. The significantly high occurrence of 1p36 breakpoint in structural rearrangements and its involvement in recurrent translocations suggest that the region is bearing gene(s) that are important in lymphomagenesis. Our study also showed that cytogenetically evident deletions were frequent in chromosome 1p, almost always involving the p36 region, whereas duplications were rare and never encompassed the p36 region. Chromosome band 1p36 harbors many candidate tumor suppressor genes, and we propose that one or more of these genes might be deleted or functionally disrupted as a molecular consequence of the rearrangements, thus contributing to lymphomagenesis.

Immortalized human corneal epithelial cells for ocular toxicity and inflammation studies.

PURPOSE: To develop a metabolically competent, human immortalized corneal epithelial cell line for use in toxicity and inflammation studies. METHODS: Primary corneal epithelial cells (P-CEPI) were immortalized by a recombinant simian virus (SV)40 T antigen retroviral vector defective for viral replication. The cells were grown in serum-free medium with the addition of bovine pituitary extract, cloned at passage 15 and one of the best-growing clones, CEPI-17-CL4, was extensively characterized for differentiation and metabolic characteristics of the human corneal epithelium. Methods used were immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis, and enzyme assays. RESULTS: The CEPI-17-CL4 cells showed a typical cobblestone morphology, grew to more than 200 passages and expressed the SV40 T antigen in the nucleus of every cell. Immunofluorescence staining for CEPI-17-CL4 cells was strongly positive for keratins (K)8, K18, and K19 and vimentin; weakly positive for K3, K13, and K17; and negative for K4, K7, and K14. Expression of cytokines (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and IL-ra), growth factors (transforming growth factor [TGF]-alpha, epidermal growth factors [EGF], EGF receptor [EGFR], TGF-beta1, TGF-beta2, and platelet-derived growth factor-beta) and cytochrome P450 enzymes (1A1, 2C, 2E1, and 3A5) was similar in CEPI-17-CL4 cells and human corneal epithelial samples obtained in biopsy. The CEPI-17-CL4 cells were metabolically competent for enzymes glutathione S-transferase, quinone reductase, aflatoxin aldehyde reductase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase. CONCLUSIONS: The CEPI-17-CL4 cells are truly immortal and express an extensive array of cytokines, growth factors, and metabolic enzymes that resemble the original tissue. These characteristics, which remain stable up to high passage, will allow reproducible, mechanistic studies on toxicity, inflammation, and wound healing.