Activation Of Normal Fibroblasts Research Articles

Colorectal Cancer Bioengineered Microtissues as a Model to Replicate Tumor-ECM Crosstalk and Assess Drug Delivery Systems In Vitro.

Current 3D cancer models (in vitro) fail to reproduce complex cancer cell extracellular matrices (ECMs) and the interrelationships occurring (in vivo) in the tumor microenvironment (TME). Herein, we propose 3D in vitro colorectal cancer microtissues (3D CRC μTs), which reproduce the TME more faithfully in vitro. Normal human fibroblasts were seeded onto porous biodegradable gelatin microbeads (GPMs) and were continuously induced to synthesize and assemble their own ECMs (3D Stroma μTs) in a spinner flask bioreactor. Then, human colon cancer cells were dynamically seeded onto the 3D Stroma μTs to achieve the 3D CRC μTs. Morphological characterization of the 3D CRC μTs was performed to assess the presence of different complex macromolecular components that feature in vivo in the ECM. The results showed the 3D CRC μTs recapitulated the TME in terms of ECM remodeling, cell growth, and the activation of normal fibroblasts toward an activated phenotype. Then, the microtissues were assessed as a drug screening platform by evaluating the effect of 5-Fluorouracil (5-FU), curcumin-loaded nanoemulsions (CT-NE-Curc), and the combination of the two. When taken together, the results showed that our microtissues are promising in that they can help clarify complex cancer-ECM interactions and evaluate the efficacy of therapies. Moreover, they may be combined with tissue-on-chip technologies aimed at addressing further studies in cancer progression and drug discovery.

Exosomal microRNAs synergistically trigger stromal fibroblasts in breast cancer

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC progression is sustained by recruitment of a strong tumor microenvironment (TME) mainly composed of cancer-associated fibroblasts (CAFs) able to endorse tumor hallmarks. Increasing evidences demonstrate that exosomes mediate the crosstalk between cancer cells and the TME. We examined TNBC-derived exosomes and their microRNA (miRNA) cargo in activation of normal fibroblasts (NFs) toward CAFs. We demonstrated that TNBC cell-derived exosomes increased NF collagen contraction and migration alongside CAF molecular markers. Exosome-activated fibroblasts promoted the invasion potential of normal breast epithelial cells, as assessed by an organotypic co-culture assay that resembled the in vivo context. We also investigated TNBC cell-derived exosome cargo in activating NFs to CAFs by performing small RNA sequencing. We found that the synergistic action of miR-185-5p, miR-652-5p, and miR-1246 boosted fibroblast migration and contraction, promoting specific CAF subspecialization toward a pro-migratory functional state. These data highlight the role of breast cancer cells in re-education of the TME and their contribution to tumor evolution.

The Interaction Between Long Non-Coding RNAs and Cancer-Associated Fibroblasts in Lung Cancer.

Despite great advances in research and treatment, lung cancer is still one of the most leading causes of cancer-related deaths worldwide. Evidence is mounting that dynamic communication network in the tumor microenvironment (TME) play an integral role in tumor initiation and development. Cancer-associated fibroblasts (CAFs), which promote tumor growth and metastasis, are the most important stroma component in the tumor microenvironment. Consequently, in-depth identification of relevant molecular mechanisms and biomarkers related to CAFs will increase understanding of tumor development process, which is of great significance for precise treatment of lung cancer. With the development of sequencing technologies such as microarray and next-generation sequencing, lncRNAs without protein-coding ability have been found to act as communicators between tumor cells and CAFs. LncRNAs participate in the activation of normal fibroblasts (NFs) to CAFs. Moreover, activated CAFs can influence the gene expression and secretion characteristics of cells through lncRNAs, enhancing the malignant biological process in tumor cells. In addition, lncRNA-loaded exosomes are considered to be another important form of crosstalk between tumor cells and CAFs. In this review, we focus on the interaction between tumor cells and CAFs mediated by lncRNAs in the lung cancer microenvironment, and discuss the analysis of biological function and molecular mechanism. Furthermore, it contributes to paving a novel direction for the clinical treatment of lung cancer.

Albumin Conjugates of Thiosemicarbazone and Imidazole-2-thione Prochelators: Iron Coordination and Antiproliferative Activity.

The central role of iron in tumor progression and metastasis motivates the development of iron-binding approaches in cancer chemotherapy. Disulfide-based prochelators are reductively activated upon cellular uptake to liberate thiol chelators responsible for iron sequestration. Herein, a trimethyl thiosemicarbazone moiety and the imidazole-2-thione heterocycle are incorporated in this prochelator design. Iron binding of the corresponding tridentate chelators leads to the stabilization of a low-spin ferric center in 2 : 1 ligand-to-metal complexes. Native mass spectrometry experiments show that the prochelators form stable disulfide conjugates with bovine serum albumin, thus affording novel bioconjugate prochelator systems. Antiproliferative activities at sub-micromolar levels are recorded in a panel of breast, ovarian and colorectal cancer cells, along with significantly lower activity in normal fibroblasts.

Expression of EMT-Related Genes in Hybrid E/M Colorectal Cancer Cells Determines Fibroblast Activation and Collagen Remodeling.

Collagen, the main non-cellular component of the extracellular matrix (ECM), is profoundly reorganized during tumorigenesis and has a strong impact on tumor behavior. The main source of collagen in tumors is cancer-associated fibroblasts. Cancer cells can also participate in the synthesis of ECM; however, the contribution of both types of cells to collagen rearrangements during the tumor progression is far from being clear. Here, we investigated the processes of collagen biosynthesis and remodeling in parallel with the transcriptome changes during cancer cells and fibroblasts interactions. Combining immunofluorescence, RNA sequencing, and second harmonic generation microscopy, we have explored the relationships between the ratio of epithelial (E) and mesenchymal (M) components of hybrid E/M cancer cells, their ability to activate fibroblasts, and the contributions of both cell types to collagen remodeling. To this end, we studied (i) co-cultures of colorectal cancer cells and normal fibroblasts in a collagen matrix, (ii) patient-derived cancer-associated fibroblasts, and (iii) mouse xenograft models. We found that the activation of normal fibroblasts that form dense collagen networks consisting of large, highly oriented fibers depends on the difference in E/M ratio in the cancer cells. The more-epithelial cells activate the fibroblasts more strongly, which correlates with a dense and highly ordered collagen structure in tumors in vivo. The more-mesenchymal cells activate the fibroblasts to a lesser degree; on the other hand, this cell line has a higher innate collagen remodeling capacity. Normal fibroblasts activated by cancer cells contribute to the organization of the extracellular matrix in a way that is favorable for migratory potency. At the same time, in co-culture with epithelial cancer cells, the contribution of fibroblasts to the reorganization of ECM is more pronounced. Therefore, one can expect that targeting the ability of epithelial cancer cells to activate normal fibroblasts may provide a new anticancer therapeutic strategy.

Three-dimensional tumor model mimics stromal - breast cancer cells signaling.

Tumor stroma is a major contributor to the biological aggressiveness of cancer cells. Cancer cells induce activation of normal fibroblasts to carcinoma-associated fibroblasts (CAFs), which promote survival, proliferation, metastasis, and drug resistance of cancer cells. A better understanding of these interactions could lead to new, targeted therapies for cancers with limited treatment options, such as triple negative breast cancer (TNBC). To overcome limitations of standard monolayer cell cultures and xenograft models that lack tumor complexity and/or human stroma, we have developed a high throughput tumor spheroid technology utilizing a polymeric aqueous two-phase system to conveniently model interactions of CAFs and TNBC cells and quantify effects on signaling and drug resistance of cancer cells. We focused on signaling by chemokine CXCL12, a hallmark molecule secreted by CAFs, and receptor CXCR4, a driver of tumor progression and metastasis in TNBC. Using three-dimensional stromal-TNBC cells cultures, we demonstrate that CXCL12 – CXCR4 signaling significantly increases growth of TNBC cells and drug resistance through activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways. Despite resistance to standard chemotherapy, upregulation of MAPK and PI3K signaling sensitizes TNBC cells in co-culture spheroids to specific inhibitors of these kinase pathways. Furthermore, disrupting CXCL12 – CXCR4 signaling diminishes drug resistance of TNBC cells in co-culture spheroid models. This work illustrates the capability to identify mechanisms of drug resistance and overcome them using our engineered model of tumor-stromal interactions.

Epigenetic Silencing of Lysyl Oxidase-Like-1 through DNA Hypermethylation in an Autosomal Recessive Cutis Laxa Case

We have recently reported a case of cutis laxa caused by a fibulin-5 missense mutation (p.C217R). Skin fibroblasts from this individual showed an abnormal pattern of expression of several genes coding for elastic fiber-related proteins, including lysyl oxidase-like-1 (LOXL1). In this study we intended to elucidate the mechanism responsible for LOXL1 downregulation in these fibulin-5-mutant cells. We identified a proximal region (-442/-342) of the human LOXL1 promoter in which two binding sites for the transcription factor specific protein 1 (Sp-1) are required for gene activity in normal fibroblasts. Binding of Sp-1 to these sequences was dramatically reduced within cutis laxa cells, although Sp-1 expression was normal. Further analysis of the promoter sequence found increased methylation levels in cutis laxa cells compared with cells from unaffected individuals. When DNA methyltransferase activity was transiently inhibited in cutis laxa cells using the 5-aza-2'-deoxycytidine, we found a significant increase in LOXL1 expression. In conclusion, besides changes caused by the fibulin-5 mutation, LOXL1 gene regulation is affected by an epigenetic mechanism that can be reversed by an inhibitor of DNA methyltransferase activity. It is not yet known whether LOXL1 gene expression is affected in all cases of cutis laxa arising from fibulin-5 mutation.

AP-1 overexpression impairs corticosteroid inhibition of collagen production by fibroblasts isolated from asthmatic subjects

Asthma is characterized by airway remodeling associated with an increase in the deposition of ECM proteins such as type I collagen. These components are mainly produced by fibroblasts. Inhaled corticosteroids are considered the cornerstone of asthma therapy. Despite substantial evidence as to the anti-inflammatory action of corticosteroids, their effect on controlling ECM protein deposition in the airways is not completely understood. This study determined the effect of dexamethasone (Dex) on collagen production by bronchial fibroblasts derived from asthmatic and healthy subjects. Expression of procollagen mRNA in fibroblasts from asthmatics and normal controls was determined by quantitative PCR. Regulation of the procollagen-alpha(1)I promoter was evaluated by transient transfections. Transforming growth factor-beta (TGF-beta) protein expression was determined by ELISA. Protein expression of glucocorticoid receptor (GR) and interaction with activator protein-1 (AP-1), a collagen regulatory transcription factor, was assessed by Western blots, coimmunoprecipitations, and EMSA. AP-1 overexpression was performed by transient transfection using c-Fos/c-Jun expression plasmids. Dex significantly downregulated procollagen production and promoter activity in normal fibroblasts but had no effect on asthmatic fibroblasts. AP-1 and GR interaction increased after Dex stimulation in asthmatic fibroblasts. AP-1 overexpression in control fibroblasts abrogated collagen gene response to Dex. These results show that Dex failed to reduce collagen production in fibroblasts from asthmatic subjects. This impaired response may be related to AP-1 overexpression in these cells.

Gene therapy for pretreated ovarian cancer via a novel fiber modified mdr1 targeting conditionally replicating adenovirus

e16513 Background: Ovarian cancer (OC) is a leading cause of death from gynecologic malignancies. Oncolytic adenoviruses (CRAd) are a new approach for cancer treatment. In this study, we constructed a fiber-modified CRAd containing the multi drug resistance gene (mdr) 1 promoter to control viral replication (Ad5/3MDR1). Antineoplastic agents induce mdr1-gene expression triggering chemoresistance. Methods: RT-PCR was performed for expression of mdr1 in cell lines and primary patient samples. We constructed Ad5/3MDR1luc1 to determine relative activity in a variety of cell lines, patient samples, and normal control cells. Replication and oncolysis of Ad5/3MDR1 were assessed in naïve and pretreated OC cell lines. The specificity of viral replication was analyzed in co-culture of pretreated OC cell lines and human fibroid cells. A mouse model of peritoneal carcinomatosis was used to evaluate the efficacy of a combined chemo- and gene therapy for OC. Results: RT-PCR reveals a significantly (p < 0.05) increased expression of the mdr1 gene in OC cell lines and patient samples. Compared to the ubiquitous cmv promoter, mdr1 showed a high level of activity in chemoresistant OC cell lines (7.3%-11.5 %) and OC patient samples (8.8%-12.4%), whereas activity in normal fibroblasts (<1%) was low. Ad5/3MDR1 shows highly selective replication in chemoresistant OC cells in a co-culture with fibroblast cells. Cell killing of Ad5/3MDR1 was comparable to Ad5/3Δ24, a CRAD that replicates in cancer cells inactive in the Rb/p16 pathway, in chemotherapy naïve OC cells but displayed significantly (1.5 log; p < 0.05) higher oncolytic potency in pretreated OC cell lines and primary cells from pretreated OC patients. Moreover, in a therapeutic orthotopic mouse model of peritoneal carcinomatosis, dramatically enhanced survival was noted with Ad5/3MDR1. Conclusions: Ad5/3MDR1 is a promising candidate for gene therapy of metastatic chemoresistant OC with great potential for clinical testing. No significant financial relationships to disclose.

RESOLUTION OF BONE MARROW FIBROSIS IN A PATIENT TREATED FOR PROMYELOCYTIC LEUKEMIA.

Introduction Bone marrow fibrosis is known to be associated with acute myeloid leukemia (AML), most frequently with AML-M7. However, it is rarely seen with acute promyelocytic leukemia (APML). The fibrosis is thought to occur secondary to activation of normal fibroblasts by cytokines secreted by the leukemia cells and often can be reversed with therapy. The overall frequency of fibrosis in patients with APML is unknown. Whether there is a prognostic implication is unknown as well. We report the case of a patient with bone marrow reticulin fibrosis at presentation with a diagnosis of APML that improved with therapy directed at the leukemia. Case Report A 42-year-old female presented to the emergency room with progressive weakness and fatigue of 1 month9s duration. She had no fever, chills, or bruising. She was on no prior medications but had not seen a doctor in over 5 years. Bloodwork was performed, and complete blood count showed a WBC count of 9.11 × 10ˆ3/μL, hemoglobin was 3.9 g/dL, and platelets were 15,000/μL with 33% blasts seen on peripheral smear. Bone marrow aspirate and biopsy were performed. The marrow showed blasts comprised > 90% of all cellular elements, with 50% of the marrow space being fibrotic. Reticulin was markedly increased. The blasts had folded nuclei and scant cytoplasmic granules. Findings were morphologically consistent with M3, a microgranular variant of the FAB classification of AML. Molecular analysis for t(15;17) by fluorescence in situ hybridization analysis was positive in the bone marrow and peripheral blood. Patient was initiated on induction therapy with all-trans retinoic acid (ATRA) and idarubicin. Repeat bone marrow biopsy in 2 months showed a complete remission (CR), with improvement in fibrosis from severe to moderate. She was then treated with two cycles of consolidation therapy with idarubicin. A third bone marrow biopsy continued to show CR pattern with further improvemen in reticulin fibrosis to minimal. The patient began maintenance therapy with daily 6-mercaptopurine, weekly methotrexate, and ATRA daily for 15 days monthly. Conclusion Bone marrow reticulin fibrosis is commonly associated with AML but rarely seen with APML. This case highlights that, although rare, it may be a secondary process of the leukemia and that therapy directed at the leukemia can help the fibrosis process.

Constitutively phosphorylated Smad3 interacts with Sp1 and p300 in scleroderma fibroblasts

To elucidate the role of transforming growth factor-beta (TGF-beta)/Smad signalling in the increased expression of the collagen gene in systemic sclerosis (SSc) fibroblasts. Dermal fibroblasts from seven patients with diffuse SSc of recent onset and from seven healthy individuals were studied. The expression levels of Smad2, Smad3 and Smad4 proteins were determined by immunoblotting. Smad3 phosphorylation and the interaction of Smad3 with Sp1 or p300 were analysed using immunoprecipitation. The effects of overexpression of Smad proteins or Sp1 on the human alpha2(I) collagen gene transcription were investigated with chloramphenicol acetyltransferase (CAT) assays using the -772 COL1A2/CAT construct. Constitutive increased Smad3 phosphorylation was detected in SSc fibroblasts compared with normal fibroblasts. Increased interaction of Smad3 with Sp1 as well as p300 was also detected in SSc fibroblasts. The overexpression of Smad3 caused an increase of up to 5-fold in COL1A2 promoter activity in normal fibroblasts, while Smad3 caused a small increase in COL1A2 promoter activity in SSc fibroblasts. However, neither Smad2 nor Smad4 caused significant effects in COL1A2 promoter activity in normal fibroblasts or SSc fibroblasts. The overexpression of Sp1 caused further increase in COL1A2 promoter activity stimulated by TGF-beta in normal fibroblasts, but did not change COL1A2 promoter activity in the presence of TGF-beta in SSc fibroblasts. The combined overexpression of Smad3 and Sp1 significantly enhanced TGF-beta response in normal fibroblasts, but less markedly in SSc fibroblasts. These results suggested that SSc fibroblasts are less sensitive to exogenous TGF-beta stimulation because they are already activated by the autocrine TGF-beta loop.

Differential effects of the immunosuppressant FK‐506 on human α2(I) collagen gene expression and transforming growth factor β signaling in normal and scleroderma fibroblasts

To investigate the effects of FK-506 on the expression of the human alpha2(I) collagen gene and transforming growth factor beta (TGFbeta) signaling in normal and scleroderma fibroblasts. The expression levels of type I procollagen protein and alpha2(I) collagen messenger RNA (mRNA) were analyzed by immunoblotting and Northern blotting, respectively. The promoter activities of alpha2(I) collagen gene and 3TP-Lux were determined by transient transfection assay. Interaction between TGFbeta receptor type I and FK-506 binding protein 12 (FKBP12) was evaluated by immunoprecipitation. FK-506 did not affect the basal expression of type I procollagen protein or alpha2(I) collagen mRNA, but it significantly reduced the TGFbeta1-induced expression of type I procollagen protein and alpha2(I) collagen mRNA in normal fibroblasts. The effect of FK-506 was regulated posttranscriptionally, but not transcriptionally. In scleroderma fibroblasts, FK-506 significantly reduced the expression of type I procollagen protein and alpha2(I) collagen mRNA through posttranscriptional regulation, but not transcriptional regulation. FK-506 increased the basal activity of the 3TP-Lux promoter, but it did not affect the TGFbeta1-induced promoter activity in normal fibroblasts. In contrast, FK-506 did not affect the basal or the TGFbeta1-induced 3TP-Lux promoter activity in scleroderma fibroblasts. Furthermore, FKBP12, which protects TGFbeta receptor type I from ligand-independent activation by TGFbeta receptor type II, constitutively dissociated from TGFbeta receptor type I in scleroderma fibroblasts. FK-506 inhibits alpha2(I) collagen gene expression by reducing the stability of mRNA without exhibiting its activation effect on TGFbeta signaling in scleroderma fibroblasts.

Disruption of transforming growth factor beta signaling and profibrotic responses in normal skin fibroblasts by peroxisome proliferator-activated receptor gamma.

In fibroblasts, transforming growth factor beta (TGF beta) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGF beta-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGF beta/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) in normal skin fibroblasts and its effect on TGF beta-induced cellular responses. The expression and activity of PPAR gamma in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPAR gamma activation by naturally occurring and synthetic ligands on collagen synthesis and alpha-smooth muscle actin (alpha-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPAR gamma expression constructs. The PPAR gamma receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPAR gamma resulted in modest suppression of basal collagen gene expression, it abrogated TGF beta-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPAR gamma in fibroblasts, and was blocked by a selective antagonist of PPAR gamma signaling or by transfection of fibroblasts with dominant-negative PPAR gamma constructs. Furthermore, PPAR gamma ligands abrogated TGF beta-induced expression of alpha-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGF beta was suppressed by PPAR gamma despite the absence of a consensus PPAR gamma-response element in the targeted promoters. Ligand-induced activation of fibroblast PPAR gamma had no effect on protein expression of cellular Smad3 or Smad7. By abrogating of TGF beta-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPAR gamma may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPAR gamma activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.

Expression of cathepsin L and its endogenous inhibitors in immortal and transformed fibroblasts

Methods: The activities of cathepsin L and its endogenous inhibitors were analyzed in rat embryo fibroblasts, immortalized and transformed by different genes. Results: Regardless of the transfecting agent used (DNA of adenovirus SA7 or polyomavirus LT gene), the immortal cells showed an increase in the cathepsin L activity (in both lysates and conditioned media) compared to primary fibroblasts. Transformed cells exhibited either an increase (with c-Ha-ras gene) or decrease (with E7 HPV gene) in cathepsin L activity in lysates as opposed to immortal cells. Conclusions: The data are suggestive of alterations in the trafficking of cathepsin L upon fibroblast transfection with polyomavirus LT gene and E7 HPV gene. An endogenous inhibitor(s) of cysteine proteinase was found in conditioned media, but not in lysates, of all cell cultures studied and its activity in normal fibroblasts was higher than in media of immortal and transformed cells.

The control of ccn2* (ctgf) gene expression in normal and scleroderma fibroblasts

Although the role of transforming growth factor β (TGFβ) in initiating fibrosis is well established, the role that TGFβ plays in maintaining fibrosis is unclear. The gene encoding connective tissue...

CTGF and SMADs, Maintenance of Scleroderma Phenotype Is Independent of SMAD Signaling

In normal adult fibroblasts, transforming growth factor-beta (TGFbeta) induces the expression of connective tissue growth factor (CTGF). CTGF independently promotes fibroblast proliferation and matrix deposition, and in acute models of fibrosis promotes cell proliferation and collagen deposition acting synergistically with TGFbeta. In contrast to normal fibroblasts, fibroblasts cultured from fibrotic tissues express high basal levels of CTGF, even in the absence of added TGFbeta. Induction of transcription by TGFbeta requires the action of SMAD proteins. In this report we have investigated the role of SMADs in the TGFbeta-induction of CTGF in normal fibroblasts and in the elevated levels of CTGF expression found in dermal fibroblasts cultured from lesional areas of patients with scleroderma, a progressive fibrotic disorder that can affect all organs of the body. We have identified a functional SMAD binding site in the CTGF promoter. TGFbeta-induction of CTGF is dependent on SMAD3 and SMAD4 but not SMAD2 and is p300-independent. However, mutation of the SMAD binding site does not reduce the high level of CTGF promoter activity observed in dermal fibroblasts cultured from lesional areas of scleroderma patients. Conversely, the previously termed TGFbetaRE in the CTGF promoter is required for basal CTGF promoter activity in normal fibroblasts and for the elevated level of CTGF promoter activity in scleroderma fibroblasts. Thus, the maintenance of the fibrotic phenotype in scleroderma fibroblasts, as visualized by excess CTGF expression, appears to be independent of SMAD-dependent TGFbeta signaling. Furthermore, given CTGF's activities, the high level of CTGF expression observed in scleroderma lesions may contribute to the excessive scarring observed in this disorder.

Congenital Erythropoietic Porphyria: Prolonged High-Level Expression and Correction of the Heme Biosynthetic Defect by Retroviral-Mediated Gene Transfer into Porphyric and Erythroid Cells

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder resulting from the deficient activity of the heme biosynthetic enzyme uroporphyrinogen III synthase (UROS). Severely affected patients are transfusion dependent and have mutilating cutaneous manifestations. Successful bone marrow transplantation has proven curative, providing the rationale for stem cell gene therapy. Toward this goal, two retroviral MFG vectors containing the UROS cDNA were constructed, one with the wild-type sequence (MFG-UROS-wt) and a second with an optimized Kozak consensus sequence (MFG-UROS-K). Following transduction of CEP fibroblasts, the MFG-UROS-wt and MFG-UROS-K vectors increased the endogenous activity without selection to levels that were 18- and 5-fold greater, respectively, than the mean activity in normal fibroblasts. Notably, the MFG-UROS-wt vector expressed UROS activity in CEP fibroblasts at these high levels for over 6 months without cell toxicity. Addition of either δ-aminolevulinic acid (ALA) or ferric chloride did not affect expression of the transduced UROS gene nor did the increased concentrations of uroporphyrin isomers or porphyrin intermediates affect cell viability. Similarly, transduction of CEP lymphoblasts with the MFG-UROS-wt vector without G418 selection increased the endogenous UROS activity by 7-fold or almost 2-fold greater than that in normal lymphoblasts. Transduction of K562 erythroleukemia cells by cocultivation with the MFG-UROS-wt producer cells increased their high endogenous UROS activity by 1.6-fold without selection. Clonally isolated K562 cells expressed UROS for over 4 months at mean levels 4.7-fold greater than the endogenous activity without cell toxicity. Thus, the prolonged, high-level expression of UROS in transduced CEP fibroblasts and lymphoblasts, as well as in transduced K562 erythroid cells, demonstrated that the enzymatic defect in CEP cells could be corrected by retroviral-mediated gene therapy without selection and that the increased intracellular porphyrin intermediates were not toxic to these cells, even when porphyrin production was stimulated by supplemental ALA or iron. Thesein vitrostudies provide the rationale forex vivostem cell gene therapy in severely affected patients with CEP.

The adenovirus-mediated delivery of a reporter gene permits the assessment of androgen receptor function in genital skin fibroblast cultures. Stimulation of Gs and inhibition of G(o).

Defects in the androgen receptor cause a spectrum of abnormalities in genetic males ranging from phenotypic women with testicular feminization to men with minor defects in fertility and/or virilization. The diagnosis of androgen resistance can be quite cumbersome, including analysis of the family history, karyotyping, endocrine studies, measurement of androgen binding in genital skin fibroblasts, and, in some instances, sequencing of mutant cDNAs. Furthermore, androgen-binding studies may be normal in patients with qualitative receptor abnormalities or mutations in the DNA-binding domain of the receptor. To circumvent these difficulties, we have used a recombinant adenovirus to deliver an androgen-responsive reporter gene (mouse mammary tumor virus-luciferase) to fibroblasts cultured from genital skin from 12 normal controls and from eight individuals with complete testicular feminization. Following incubation with androgen (2 nM mibolerone) for 72 h, luciferase activity in normal fibroblasts increased > 10-fold (range 11-200-fold) in a manner that corresponded with the level of androgen receptor detected in ligand-binding assays. By contrast, luciferase activity increased negligibly in fibroblasts from individuals with testicular feminization (average = 1.2-fold increase). This assay permits a direct assessment of endogenous androgen receptor function in cells and should be a powerful aid in the diagnosis of androgen resistance.

Androgen dependent stimulation of aromatase activity in genital skin fibroblasts from normals and patients with androgen insensitivity

To measure the effect of androgens or aromatase activity as an index of androgen responsiveness in patients with androgen insensitivity. Genital skin fibroblasts were established in culture using primary skin explants obtained from normal males at the time of circumcision and from androgen insensitive patients who had surgery either for gonadectomy (complete androgen insensitivity syndrome) or for reconstruction of the external genitalia (partial androgen insensitivity syndrome). Foreskin samples were obtained at the time of circumcision in 27 normal males. Scrotal or labia majora skin was obtained at the time of surgery from 14 patients with the complete and 22 with the partial forms of the androgen insensitivity syndrome. Basal and stimulated levels of aromatase activity were measured in genital skin fibroblasts following preincubation with natural and synthetic, nonmetabolizable androgens. Following a 48-hour preincubation with testosterone or dihydrotestosterone, there was a five to six-fold stimulation of aromatase activity in normal fibroblasts. Mibolerone, a synthetic androgen, produced similar results. The stimulatory effect was blocked by anti-androgens. Seven patients with partial androgen insensitivity, of whom four were either receptor deficient or showed a qualitative defect in androgen binding, had reduced mibolerone induced stimulation of aromatase activity. All ten patients with receptor negative complete androgen insensitivity had an absent response. There was no aromatase induction in a further three patients with complete androgen insensitivity who were receptor positive. Two siblings in the latter group had an exon deletion encoding for part of the DNA binding domain of the androgen receptor. Androgens stimulate aromatase activity in genital skin fibroblasts from normals. The response is mediated via the androgen receptor and can be decreased or absent in patients with the androgen insensitivity syndrome. This may be a useful in-vitro marker of androgen responsiveness in such patients.

Calcium antagonists and low density lipoprotein receptors.

The effect of different calcium antagonists on receptor-mediated LDL catabolism by human cells in culture was investigated. The calcium antagonists have been recently classified in six types, based on their pharmacological activities. The three types selective for the slow calcium channels (types I, II, and III), and the nonselective type IV have been investigated in respect to LDL metabolism. Calcium antagonists of type I (verapamil-related compounds) and type III (diltiazem) induce an increase of receptor-mediated uptake of human LDL. In contrast, calcium antagonists of type II (nifedipine-related compounds) and type IV (flunarizine) are inactive. Verapamil and diltiazem stimulate LDL receptor activity in normal fibroblasts, in fibroblasts obtained from a hypercholesterolemic type IIa heterozygous patient, in the human hepatoma cell line HepG2, but not in receptor-negative cells. The stimulatory effect depends on drug concentrations in the culture medium. Cycloheximide and alpha-amanitin prevent the stimulating effect of calcium antagonists on LDL uptake. The possible mechanisms of this action of calcium antagonists and the relationship between the in vitro stimulation of LDL receptor activity and the in vivo inhibition of lipid deposition in the arterial wall elicited by calcium antagonists are discussed. Calcium antagonists may exert part of their antiatherosclerotic activity by counteracting the inhibition of receptor-mediated lipid metabolism induced by calcium deposition in the cellular components of the arterial walls.