Immortalization Of Fibroblasts Research Articles

Local injection of CCL19-expressing mesenchymal stem cells augments the therapeutic efficacy of anti-PD-L1 antibody by promoting infiltration of immune cells.

BackgroundMesenchymal stem/stromal cells (MSC) accumulate and reside in tumor sites.MethodsTaking advantage of this feature in anticancer therapy, immortalized murine MSC (iMSC) were genetically altered to produce chemokine (C-C motif) ligand 19 (iMSC/CCL19), which attracts dendritic cells (DC) and T lymphocytes. Thereafter, iMSC/CCL19 were examined for their therapeutic efficacy using a syngeneic CT26 colon carcinoma cell line.ResultsCo-injection of iMSC/CCL19 into mice significantly suppressed the in vivo growth of CT26 cells compared with that of CCL19-expressing immortalized fibroblasts (iFib/CCL19). This anticancer effect was not observed when injected in CT26-bearing nude mice. Co-injected iMSC/CCL19 survived longer than iFib/CCL19 in the tumor sites. In a therapeutic model, local injection of iMSC/CCL19 suppressed the tumor growth, and increased IFN (interferon)-γ+ CD8+ T cells and CCR7+ DC infiltration in tumor site was observed when treated with iMSC/CCL19, but not with iMSC. This antitumor effect was completely negated by depletion of CD4+ cells and partially negated by depletion of CD8+ cells. Furthermore, the antitumor effects induced by local injection of iMSC/CCL19 were augmented by additional therapy with anti-programmed death (PD)-ligand 1 (PD-L1) antibody, but not with anti-PD-1 antibody. This combination therapy cured most of the tumors in CT26-bearing mice.ConclusionThese results suggest that local therapy with iMSC/CCL19 can suppress tumor growth via effective recruitment of CCR7+ DC into tumor sites and increase IFN-γ+ CD8+ T cells, and that combination with anti-PD-L1 antibody therapy can be a powerful anticancer therapy.

Novel compound heterozygous pathogenic variants in nucleotide-binding protein like protein (NUBPL) cause leukoencephalopathy with multi-systemic involvement

NUBPL (Nucleotide-binding protein like) protein encodes a member of the Mrp/NBP35 ATP-binding proteins family, deemed to be involved in mammalian complex I (CI) assembly process. Exome sequencing of a patient presenting with infantile-onset hepatopathy, renal tubular acidosis, developmental delay, short stature, leukoencephalopathy with minimal cerebellar involvement and multiple OXPHOS deficiencies revealed the presence of two novel pathogenic compound heterozygous variants in NUBPL (p.Phe242Leu/p.Leu104Pro). We investigated patient's and control immortalised fibroblasts and demonstrated that both the peripheral and the membrane arms of complex I are undetectable in mutant NUBPL cells, resulting in virtually absent CI holocomplex and loss of enzyme activity. In addition, complex III stability was moderately affected as well. Lentiviral-mediated expression of the wild-type NUBPL cDNA rescued both CI and CIII assembly defects, confirming the pathogenicity of the variants. In conclusion, this is the first report describing a complex multisystemic disorder due to NUBPL defect. In addition, we confirmed the role of NUBPL in Complex I assembly associated with secondary effect on Complex III stability and we demonstrated a defect of mtDNA-related translation which suggests a potential additional role of NUBPL in mtDNA expression.

A Brief History of Mitochondrial Pathologies.

The history of “mitochondrial pathologies”, namely genetic pathologies affecting mitochondrial metabolism because of mutations in nuclear DNA-encoded genes for proteins active inside mitochondria or mutations in mitochondrial DNA-encoded genes, began in 1988. In that year, two different groups of researchers discovered, respectively, large-scale single deletions of mitochondrial DNA (mtDNA) in muscle biopsies from patients with “mitochondrial myopathies” and a point mutation in the mtDNA gene for subunit 4 of NADH dehydrogenase (MTND4), associated with maternally inherited Leber’s hereditary optic neuropathy (LHON). Henceforth, a novel conceptual “mitochondrial genetics”, separate from mendelian genetics, arose, based on three features of mtDNA: (1) polyplasmy; (2) maternal inheritance; and (3) mitotic segregation. Diagnosis of mtDNA-related diseases became possible through genetic analysis and experimental approaches involving histochemical staining of muscle or brain sections, single-fiber polymerase chain reaction (PCR) of mtDNA, and the creation of patient-derived “cybrid” (cytoplasmic hybrid) immortal fibroblast cell lines. The availability of the above-mentioned techniques along with the novel sensitivity of clinicians to such disorders led to the characterization of a constantly growing number of pathologies. Here is traced a brief historical perspective on the discovery of autonomous pathogenic mtDNA mutations and on the related mendelian pathology altering mtDNA integrity.

BRCa1 participates in XIST RNa localization on inactive x chromosome

Introduction . Inactive X chromosome (Xi) is associated with noncoding XIST RNA, series of proteins and contains multiple epigenetic modifications that altogether determine a silence of the most of X-linked genes. Recently the data were obtained that tumor suppressor BRCA1 is also associated with Xi. The purpose of this study was to reveal the colocalization of BRCA1 and XIST RNA and precise spatial organization on Xi with the high resolution of confocal microscopy.Materials and methods . The object of the study is IMR90hTERT diploid immortalized fibroblast cell line. For BRCA1 and XIST RNA colocalization analysis on Xi the method of fluorescent hybridization in situ associated with immunofluorescent cell staining (immunoFISH) and confocal microscopy were used. For BRCA1 and heterochromatin protein-1 colocalization study the method of double immunofluorescent staining and common fluorescent microscopy were applied. Results . The study using confocal fluorescent microscopy with higher resolution has demonstrated at first the colocalization of BRCA1 with XIST RNA region of Xi revealed with XIST RNA probes and with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. Altogether, the data obtained suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the regulation of XIST RNA association with Xi. Moreover, according to the results of confocal microscopy, BRCA1 also colocalizes with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. This indicates a possible involvement of this protein in the replication of pericentromeric repeats in cellular chromosomes. Colocalization of BRCA1 with heterochromatin protein-1α presented in pericentromeric regions of all chromosomes supports this suggestion.Conclusions . Altogether, the data obtained in this study suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the association with noncoding inhibiting XIST RNA and in replication of heterochromatin regions.

Comparison of DNA repair in human cell lines

The study of genome repair dynamics and their variance among cell lines refines our understanding of cancer and may lead to better gene‐targeting therapies. Our study compares the genotoxic repair rates of two cell types: the immortal cervical cancer line HeLa and human dermal fibroblasts, or HDFa. We are employing the comet assay, a single cell gel electrophoresis technique, to quantify genetic damage after subjecting cells to varying doses of UV and X‐ray radiation. To form a comparison of repair rates between the cell lines, we are tracking levels of damage at specific increments of recovery time.HeLa exhibits greater genome instability than HDFa, presumably due to flawed DNA repair mechanisms. As such, the expectation is that the cell lines will show differing repair rates; however, prior observation has suggested the opposite. Our results demonstrate a similar repair dynamics between these two cell lines following X‐ray and UV‐C light exposures. An investigation into gene‐specific repair might shed light on specifics of nucleotide and base excision repair pathways in HeLa and HDFa cells.Support or Funding Information1) Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. UA is an AA/EO employer and educational institution and prohibits illegal discrimination against any individual: www.alaska.edu/titleIXcompliance/nondiscrimination.2) NASA EPSCoR, NNH15ZHA003CThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential contributions of nonmuscle myosin IIA and IIB to cytokinesis in human immortalized fibroblasts

Nonmuscle myosin II (NMII) plays an important role in cytokinesis by constricting a contractile ring. However, it is poorly understood how NMII isoforms contribute to cytokinesis in mammalian cells. Here, we investigated the roles of the two major NMII isoforms, NMIIA and NMIIB, in cytokinesis using a WI-38 VA13 cell line (human immortalized fibroblast). In this cell line, NMIIB tended to localize to the contractile ring more than NMIIA. The expression level of NMIIA affected the localization of NMIIB. Most NMIIB accumulated at the cleavage furrow in NMIIA-knockout (KO) cells, and most NMIIA was displaced from this location in exogenous NMIIB-expressing cells, indicating that NMIIB preferentially localizes to the contractile ring. Specific KO of each isoform elicited opposite effects. The rate of furrow ingression was decreased and increased in NMIIA-KO and NMIIB-KO cells, respectively. Meanwhile, the length of NMII-filament stacks in the contractile ring was increased and decreased in NMIIA-KO and NMIIB-KO cells, respectively. Moreover, NMIIA helped to maintain cortical stiffness during cytokinesis. These findings suggest that appropriate ratio of NMIIA and NMIIB in the contractile ring is important for proper cytokinesis in specific cell types. In addition, two-photon excitation spinning-disk confocal microscopy enabled us to image constriction of the contractile ring in live cells in a three-dimensional manner.

Cosmetic Formulation Based on an Açai Extract

(1) Background: Açai berry extract is known for its high content in polyphenols and thus is a promising ingredient for cosmetic antiaging formulations; (2) Methods: In this study, the açai extract was firstly evaluated for its total phenol content (Folin Ciocalteau essay) and antioxidant activity (radical scavenging activity—DPPH; radical cation scavenging capacity—ABTS; ferric reducing antioxidant capacity—FRAP). Next, the açai extract was included in an O/W formulation and again was evaluated for its polyphenol content and antioxidant capacity. The formulation was tested for general characteristics, physicochemical properties and microbial stability. The proliferative effect on human immortalized fibroblasts was evaluated by the MTT essay, while TAC assay served to confirm that fibroblasts are protected from UV irradiation. The irritant potential was verified on 20 volunteers. The study concluded with the assessment of the sensorial characteristics of the cosmetic formulation; (3) Results: The pure açai extract exhibited high polyphenol content and antioxidant activity, and these characteristics were preserved in the O/W formulation as well. The O/W cosmetic formulation proved to be stable under accelerated and normal conditions, and the preservatives were successful in challenging the resistance against microbial contamination. The mean irritant potential was zero in all volunteers, and the cosmetic formulation showed a good sensorial profile; (4) Conclusions: Açai extract is an interesting ingredient for cosmetic antiaging formulations.

SRSF5 functions as a novel oncogenic splicing factor and is upregulated by oncogene SRSF3 in oral squamous cell carcinoma

Alternative splicing of precursor messenger RNA has been increasingly associated with tumorigenesis. The serine/arginine-rich protein (SR) family plays key roles in the regulation of pre-mRNA alternative splicing. Increasing evidence has demonstrated that the SR protein family is involved in tumorigenesis. However, the functions and mechanisms of SR proteins in tumourigenesis remain largely unknown. In the present study, we discovered that serine/arginine-rich splicing factor 5 (SRSF5) is a novel oncogenic splicing factor that is overexpressed in oral squamous cell carcinoma (OSCC) tissues and cells, being crucial for OSCC cell proliferation and tumor formation. Overexpression of SRSF5 transformed immortal rodent fibroblasts to form tumors in nude mice, while downregulation of SRSF5 in oral squamous cell lines retarded cell growth, cell cycle progression, and tumor growth. The expression of SRSF5 is controlled by an autoregulation mechanism. Serine/arginine-rich splicing factor 3 (SRSF3) has been identified as an oncogene. We found that SRSF5 is a novel target of SRSF3. SRSF3 impairs the autoregulation of SRSF5 and promotes SRSF5 overexpression in cancer cells. Altogether, the present study demonstrated that SRSF5 is a novel oncogene that is upregulated by SRSF3 in OSCC cells.

Decreased argininosuccinate synthetase expression in Thai patients with cholangiocarcinoma and the effects of ADI-PEG20 treatment in CCA cell lines.

Cholangiocarcinoma (CCA) is a severe cancer with poor prognosis. The aim of the present study was to explore the expression of argininosuccinate synthetase (ASS), as well as the possibility of using pegylated arginine deiminase (ADI-PEG20) for the treatment of CCA. ASS expression was determined in CCA specimens from 40 patients in Thailand. Immunohistochemical detection of ASS and determination of the proliferative index, Ki-67, were carried out in paraffin-embedded sections of these specimens, as well as in two CCA cell lines, HuCCA and RmCCA-1, derived from CCA samples from patients in Thailand. In total, ~45% of the CCA specimens had low ASS expression, and the level of expression was significantly negatively associated with cell differentiation (P<0.05) and Ki-67 expression (P<0.05). The level of ASS expression in tumor cells was significantly lower than that in non-tumor cells (1.3-fold, P<0.05). The HuCCA cell line had significantly lower levels (P<0.05) of ASS expression at the mRNA and protein levels relative to those of normal human immortalized fibroblast cells (BJ-1). By contrast, the RmCCA-1 cell line showed no significant difference. In addition, the effects of ADI-PEG20 on growth inhibition, apoptosis and cell cycle arrest were determined in HuCCA and RmCCA-1 cells. ADI-PEG20 treatment reduced cell viability and cell proliferation in the two CCA cell lines, though it had no effect in immortalized BJ-1 cells. Furthermore, ADI-PEG20 treatment significantly increased G0/G1 cell cycle arrest in HuCCA, though not in RmCCA-1 cells. ASS silencing in the RmCCA-1 cell line significantly enhanced its sensitivity to ADI-PEG20 treatment. Results from the in vitro study demonstrated that ADI-PEG20 has antitumor activity against CCA with low ASS expression.

The effects of AICAR and rapamycin on mitochondrial function in immortalized mitochondrial DNA mutator murine embryonic fibroblasts.

ABSTRACTMitochondrial DNA mutations accumulate with age and may play a role in stem cell aging as suggested by the premature aging phenotype of mitochondrial DNA polymerase gamma (POLG) exonuclease-deficient mice. Therefore, E1A immortalized murine embryonic fibroblasts (MEFs) from POLG exonuclease-deficient and wild-type (WT) mice were constructed. Surprisingly, when some E1A immortalized MEF lines were cultured in pyruvate-containing media they slowly became addicted to the pyruvate. The POLG exonuclease-deficient MEFs were more sensitive to several mitochondrial inhibitors and showed increased reactive oxygen species (ROS) production under standard conditions. When cultured in pyruvate-containing media, POLG exonuclease-deficient MEFs showed decreased oxygen consumption compared to controls. Increased AMP-activated protein kinase (AMPK) signaling and decreased mammalian target of rapamycin (mTOR) signaling delayed aging and influenced mitochondrial function. Therefore, the effects of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, or rapamycin, an mTOR inhibitor, on measures of mitochondrial function were determined. Rapamycin treatment transiently increased respiration only in WT MEFs and, under most conditions, increased ATP levels. Short term AICAR treatment transiently increased ROS production and, under most conditions, decreased ATP levels. Chronic AICAR treatment decreased respiration and ROS production in WT MEFs. These results demonstrate the context-dependent effects of AICAR and rapamycin on mitochondrial function.

Whole chromosome loss and associated breakage-fusion-bridge cycles transform mouse tetraploid cells.

Whole chromosome gains or losses (aneuploidy) are a hallmark of ~70% of human tumors. Modeling the consequences of aneuploidy has relied on perturbing spindle assembly checkpoint (SAC) components, but interpretations of these experiments are clouded by the multiple functions of these proteins. Here, we used a Cre recombinase-mediated chromosome loss strategy to individually delete mouse chromosomes 9, 10, 12, or 14 in tetraploid immortalized murine embryonic fibroblasts. This methodology also involves the generation of a dicentric chromosome intermediate, which subsequently undergoes a series of breakage-fusion-bridge (BFB) cycles. While the aneuploid cells generally display a growth disadvantage invitro, they grow significantly better in low adherence sphere-forming conditions and three of the four lines are transformed invivo, forming large and invasive tumors in immunocompromised mice. The aneuploid cells display increased chromosomal instability and DNA damage, a mutator phenotype associated with tumorigenesis invivo Thus, these studies demonstrate a causative role for whole chromosome loss and the associated BFB-mediated instability in tumorigenesis and may shed light on the early consequences of aneuploidy in mammalian cells.

PHYTOCHEMICAL ANALYSIS, ASSESSMENT OF ANTIPROLIFERATIVE AND FREE RADICAL SCAVENGING ACTIVITY OF MORUS ALBA AND MORUS RUBRA FRUITS

Objective: Mulberry is a nontoxic commonly eaten plant, belongs to the Morus and used in folk medicine in the remedy of dysentery, antiphlogistic, diuretic, expectorant, and antidiabetic. The purpose of this study is to evaluate the antiproliferative and radical scavenging activity of the total alcoholic and successive fractions thereof of Morus alba and Morus rubra fruits. In addition, the chemical composition of the bioactive fractions of each species was investigated.Methods: The antiproliferative potential of 8 extracts on 4 human cancer cell lines, hepatocellular carcinoma (HepG2), Caucasian breast adenocarcinoma (MCF7), prostate (PC3), and colon carcinoma (HCT116) in addition to one normal cell line namely human normal immortalized skin fibroblast cells (BJ1) were carried out. Cell viability was determined using MTT assay. The potency was compared with the reference drug doxorubicin. These extracts were also assayed for 1,1-diphenyl-2-hydrazyl free radical scavenging activities. After saponification of the n-hexane fraction, unsaponifiable matter and fatty acid methyl esters were analyzed by gas liquid chromatography (GLC). The chemical composition of the bioactive fractions was investigated using gas chromatography/mass spectrometry (GC/MS) analysis.Results: All the extracts showed significant free radical scavenging activity dose-dependently. The n-hexane and dichloromethane (DCM) fractions of M. rubra exhibited potent cytotoxic activity on almost cancer cell lines. In the same pattern, ethyl acetate (EtOAc) of M. rubra has moderate cytotoxic activity against all cell lines except HepG2. DCM fraction of M. alba possessed both radical scavenging and high potential antiproliferated activities against HCT116 and MCF7 with inhibitory concentration of 43.9 and 32.3 μg/ml, respectively, while it showed no cytotoxic effect on BJ1. GLC analysis showed the major hydrocarbons in M. alba and M. rubra were heptacosane and docosane, respectively. Sterols were similar in both species but with different ratios and cholesterol was the major one. Palmitic and margaric were the major saturated fatty acid while arachidonic was the major unsaturated fatty acid in both species. GC/MS analysis showed the main compound in DCM fraction of each Morus species was palmitic acid. Furthermore, 1,11-bis-(methoxycarbonyl-ethenyl)-10,2-dihydroxy-cycloeicosane and linolelaidic acid, methyl ester were the main compounds in the EtOAc fraction of each Morus species. Whereas, the main compounds in alcoholic extract of M. alba and M. rubra were methyl-14-methyl-pentadecanoate and 1,2-O-isopropylyidene-4-nonene-1,2,3-triol, respectively.Conclusions: The results observed remarkable biological activity of the successive fractions of M. rubra more than those of M. alba and confirmed its importance as a natural bioactive source. Morus species are good candidates to be promising as possible sources for future antitumor and antioxidants in food and pharmaceutical formulations. The strong activity partly explains the potential effects of Morus species for the treatment of cancer and degenerative diseases caused by free radicals.

Cytokines, inducers and inhibitors modulate MMP-2 and MMP‑9 secretion by human Fanconi anemia immortalized fibroblasts.

Acute myeloid leukemia and head and neck squamous cell carcinomas are the major causes of mortality and morbidity in Fanconi anemia (FA) patients. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, have been implicated in tumor invasion and metastasis. Various cytokines, mitogens, growth factors, inducers and inhibitors control MMP activities. We investigated the roles of these in the regulation of MMP-2 and MMP-9 in human immortalized fibroblasts from FA. Human FA immortalized fibroblast cell lines FA-A:PD220 and FA-D2:PD20 were grown in minimum essential medium (MEM) supplemented with 15% fetal bovine serum (FBS) and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with phosphate‑buffered saline (PBS) and incubated in serum-free media with the following: phorbol 12-myristate 13-acetate (PMA) at 10-100ng/ml; tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) at 0.1-25ng/ml; lipopolysaccharide (LPS) at 10-100µg/ml; epigallocatechin gallate (EGCG) and doxycycline (Dox) at 10-100µM without and with PMA; a nutrient mixture (NM) without and with PMA at 10-1,000µg/ml; actinomycin-D and cyclohexamide at 2and4µM; retinoic acid and dexamethasone at 50µM. After 24h, media were removed and analyzed for MMP-2 and MMP-9 by zymography. Both FA cell lines expressed only MMP-2 and responded similarly to cytokines, mitogens, inducers and inhibitors. PMA potently stimulated MMP-9 and had a moderate effect on MMP-2. TNF-α showed variable effects on MMP-2 and significantly enhanced MMP-9. IL-1β enhanced MMP-2 slightly and MMP-9 significantly. LPS had a moderate stimulatory effect on MMP-2 and no effect on MMP-9. EGCG, Dox and NM, without and with PMA, downregulated MMP-2 and MMP-9 expression. Actinomycin-D, retinoic acid and dexamethasone also had inhibitory effects on MMP-2. Our results showed that cytokines, mitogens and inhibitors modulated FA fibroblast MMP-2 and MMP-9 expression, suggesting the clinical use of MMP inhibitors, particularly such potent and non-toxic ones as the NM and its component EGCG in the management of FA cancers.

The effect of two drug delivery systems in ropivacaine cytotoxicity and cytokine release by human keratinocytes and fibroblasts.

Modified drug delivery systems have been developed to improve pharmacological properties of local anaesthetics. However, the inflammatory potential of these formulations was not investigated. This study compared the in-vitro effects of ropivacaine (ropi) in plain, liposomal (MLV) or 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) formulations on cell viability, apoptosis and cytokine (IL-1α, TNF-α, IL-6 and IL-10) release. Human immortalized keratinocytes (HaCaT) and human immortalized gingival fibroblasts (HGF) were exposed to 1-100μm ropi concentrations. The cell viability was measured by XTT and LIVE/DEAD assay. Apoptosis was performed by flow cytometry, and cytokine release was measured by ELISA assay. Human immortalized keratinocyte viability was reduced by ropi and both drug delivery systems. However, none of the formulations induced apoptosis. Results showed a differential regulation of IL-1α TNF-α, IL-6 and IL-10 by HaCaT and HGF. Ropi-HP-β-CD increased twofold the IL-6 release by HGF in comparison with the control, while 100μm ropi-MLV led to an increased release of all pro-inflammatory cytokines by HGF. The loss in cell viability was not related to cellular apoptosis. Ropi complexed with HP-β-CD showed a similar cytokine release pattern when compared to the plain formulation. Thus, the HP-β-CD form was a better drug carrier than the MLV form for ropivacaine drug delivery.

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients.

The Discovery and Early Days of TGF-β: A Historical Perspective.

Transforming growth factors (TGFs) were discovered as activities that were secreted by cancer cells, and later by normal cells, and had the ability to phenotypically and reversibly transform immortalized fibroblasts. TGF-β distinguished itself from TGF-α because it did not bind to the same epidermal growth factor (EGF) receptor as TGF-α and, therefore, acted through different cell-surface receptors and signaling mediators. This review summarizes the discovery of TGF-β, the early developments in its molecular and biological characterization with its many biological activities in different cell and tissue contexts and its roles in disease, the realization that there is a family of secreted TGF-β-related proteins with many differentiation functions in development and activities in normal cell and tissue physiology, and the subsequent identification and characterization of the receptors and effectors that mediate TGF-β family signaling responses.

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results)

Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.

Cytotoxicity evaluation of large cyanobacterial strain set using selected human and murine in vitro cell models

The production of cytotoxic molecules interfering with mammalian cells is extensively reported in cyanobacteria. These compounds may have a use in pharmacological applications; however, their potential toxicity needs to be considered. We performed cytotoxicity tests of crude cyanobacterial extracts in six cell models in order to address the frequency of cyanobacterial cytotoxicity to human cells and the level of specificity to a particular cell line. A set of more than 100 cyanobacterial crude extracts isolated from soil habitats (mainly genera Nostoc and Tolypothrix) was tested by MTT test for in vitro toxicity on the hepatic and non-hepatic human cell lines HepG2 and HeLa, and three cell systems of rodent origin: Yac-1, Sp-2 and Balb/c 3T3 fibroblasts. Furthermore, a subset of the extracts was assessed for cytotoxicity against primary cultures of human hepatocytes as a model for evaluating potential hepatotoxicity. Roughly one third of cyanobacterial extracts caused cytotoxic effects (i.e. viability<75%) on human cell lines. Despite the sensitivity differences, high correlation coefficients among the inhibition values were obtained for particular cell systems. This suggests a prevailing general cytotoxic effect of extracts and their constituents. The non-transformed immortalized fibroblasts (Balb/c 3T3) and hepatic cancer line HepG2 exhibited good correlations with primary cultures of human hepatocytes. The presence of cytotoxic fractions in strongly cytotoxic extracts was confirmed by an activity-guided HPLC fractionation, and it was demonstrated that cyanobacterial cytotoxicity is caused by a mixture of components with similar hydrophobic/hydrophilic properties. The data presented here could be used in further research into in vitro testing based on human models for the toxicological monitoring of complex cyanobacterial samples.

TP53inp1 Gene Is Implicated in Early Radiation Response in Human Fibroblast Cells.

Tumor protein 53-induced nuclear protein-1 (TP53inp1) is expressed by activation via p53 and p73. The purpose of our study was to investigate the role of TP53inp1 in response of fibroblasts to ionizing radiation. γ-Ray radiation dose-dependently induces the expression of TP53inp1 in human immortalized fibroblast (F11hT) cells. Stable silencing of TP53inp1 was done via lentiviral transfection of shRNA in F11hT cells. After irradiation the clonogenic survival of TP53inp1 knockdown (F11hT-shTP) cells was compared to cells transfected with non-targeting (NT) shRNA. Radiation-induced senescence was measured by SA-β-Gal staining and autophagy was detected by Acridine Orange dye and microtubule-associated protein-1 light chain 3 (LC3B) immunostaining. The expression of TP53inp1, GDF-15, and CDKN1A and alterations in radiation induced mitochondrial DNA deletions were evaluated by qPCR. TP53inp1 was required for radiation (IR) induced maximal elevation of CDKN1A and GDF-15 expressions. Mitochondrial DNA deletions were increased and autophagy was deregulated following irradiation in the absence of TP53inp1. Finally, we showed that silencing of TP53inp1 enhances the radiation sensitivity of fibroblast cells. These data suggest functional roles for TP53inp1 in radiation-induced autophagy and survival. Taken together, we suppose that silencing of TP53inp1 leads radiation induced autophagy impairment and induces accumulation of damaged mitochondria in primary human fibroblasts.

Abstract 4024: Understanding the role of mitochondria in the progression from normal fibroblasts to DCIS associated fibroblasts

Abstract Background and Purpose: Ductal carcinoma in situ (DCIS) is a non-invasive breast cancer which may progress to invasive breast cancer. Many studies have focused on the epithelial cell mechanisms of progression from normal breast tissue to DCIS. However, in this study we want to study mechanisms of progression in cancer associated fibroblasts from normal breast tissue to DCIS. Loss of caveolin 1 (CAV1) in fibroblasts is a marker of reduced mitochondrial membrane potential and is a prognostic biomarker in ductal carcinoma in situ. High mitochondrial membrane potential in epithelial cells is a crucial determinant of carcinogenesis. Methods: In the study we use MCF10DCIS as a model for DCIS. MCF10DCIS.com (MCF10DCIS) is a clonal breast cancer cell line. The MCF10A, non-tumorigenic epithelial cell line, and human immortalized but non-transformed dermal fibroblasts (BJ1) were used study the difference between non-tumorigenic epithelial cells and DCIS and interactions between these two epithelial cells and fibroblasts. In order to study the interaction between epithelial cells and fibroblasts, we set up a coculture model system comprised both epithelial cells and fibroblasts. Immunofluorescence staining and confocal microscopy were used here to visualize the specific protein expression in different cell types. Results: (1) MCF10DCIS cells in coculture with fibroblasts have increased mitotracker red staining while as fibroblasts have decreased mitotracker staining compared to homotypic culture. However, coculture of MCF10A cells with fibroblasts did not change the mitotracker staining in either MCF10A or fibroblasts. (2) Loss of stromal CAV1 protein expression is observed in fibroblasts when fibroblasts are cocultured with MCF10DCIS cells compared to homotypic culture. CAV1 protein expression is unchanged in coculture with MCF10A cells. Conclusion: Previous studies have demonstrated that loss of stromal CAV1 and changes in mitochondrial membrane potential are factors involved in the transition from DCIS to invasive breast cancer. In this study we show that loss of stromal CAV1 and increases in epithelial mitochondrial membrane potential may play a role in the progression to DCIS. Citation Format: Ying-Hui Ko, Zhao Lin, Ubaldo E. Martinez Outschoorn. Understanding the role of mitochondria in the progression from normal fibroblasts to DCIS associated fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4024. doi:10.1158/1538-7445.AM2015-4024