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PubMed2004

Inducible short-term and stable long-term cell culture systems reveal that the PAX3-FKHR fusion oncoprotein regulates CXCR4, PAX3, and PAX7 expression.

In the pediatric cancer alveolar rhabdomyosarcoma (ARMS), the 2;13 chromosomal translocation juxtaposes the PAX3 and FKHR genes to generate a chimeric transcription factor. To explore molecular pathways altered by this oncoprotein, we generated an inducible form by fusing PAX3-FKHR to a modified estrogen receptor ligand-binding domain and expressed this construct in the RD embryonal rhabdomyosarcoma cell line. This inducible system permits short-term evaluation of downstream expression targets of PAX3-FKHR and complements a panel of stable long-term RD subclones constitutively expressing PAX3-FKHR. Using these two sets of resources, we investigated several candidate PAX3-FKHR target genes. First, we demonstrated in both short-term and long-term systems that PAX3-FKHR upregulates expression of the gene encoding the chemokine receptor CXCR4. In addition, we found that expression of wild-type PAX3 is upregulated, whereas expression of wild-type PAX7 is downregulated by PAX3-FKHR. In the presence of cycloheximide, CXCR4 and PAX3 are still inducible, supporting the hypothesis that these genes are direct transcriptional targets of PAX3-FKHR. Finally, studies of ARMS tumors revealed CXCR4, PAX3, and PAX7 expression levels consistent with our cell culture results. These findings of genes regulated by PAX3-FKHR will direct future biological and clinical investigation to important pathways contributing to ARMS tumorigenesis and progression.

Base Sequence↗
PubMed2005

Overcoming functional redundancy to elicit pachyonychia congenita-like nail lesions in transgenic mice.

Mutations affecting the coding sequence of intermediate filament (IF) proteins account for >30 disorders, including numerous skin bullous diseases, myopathies, neuropathies, and even progeria. The manipulation of IF genes in mice has been widely successful for modeling key features of such clinically distinct disorders. A notable exception is pachyonychia congenita (PC), a disorder in which the nail and other epithelial appendages are profoundly aberrant. Most cases of PC are due to mutations in one of the following keratin-encoding genes: K6, K16, and K17. Yet null alleles obliterating the function of both K6 genes (K6alpha and K6beta) or the K17 gene, as well as the targeted expression of a dominant-negative K6alpha mutant, elicit only a subset of PC-specific epithelial lesions (excluding that of the nail in mice). We show that newborn mice null for K6alpha, K6beta, and K17 exhibit severe lysis restricted to the nail bed epithelium, where all three genes are robustly expressed, providing strong evidence that this region of the nail unit is initially targeted in PC. Our findings point to significant redundancy among the multiple keratins expressed in hair and nail, which can be related to the common ancestry, clustered organization, and sequence relatedness of specific keratin genes.

Alleles↗
PubMed1996

Immortalization of pituitary cells at discrete stages of development by directed oncogenesis in transgenic mice.

Targeted expression of oncogenes in transgenic mice can immortalize specific cell types to serve as valuable cultured model systems. Utilizing promoter regions from a set of genes expressed at specific stages of differentiation in a given cell lineage, we demonstrate that targeted oncogenesis can produce cell lines representing sequential stages of development, in essence allowing both spatial and temporal immortalization. Our strategy was based on our production of a committed but immature pituitary gonadotrope cell line by directing expression of the oncogene SV40 T antigen using a gonadotrope-specific region of the human glycoprotein hormone alpha-subunit gene in transgenic mice. These cells synthesize alpha-subunit and gonadotropin-releasing hormone (GnRH) receptor, yet are not fully differentiated in that they do not synthesize the beta-subunits of luteinizing hormone (LH) or follicle-stimulating hormone (FSH). This observation lead to the hypothesis that targeting oncogenesis with promoters that are activated earlier or later in development might immortalize cells that were more primitive or more differentiated, respectively. To test this hypothesis, we used an LHbeta promoter to immortalize a cell that represents a subsequent stage of gonadotrope differentiation (expression of alpha-subunit, GnRH receptor, and LH beta-subunit but not FSH beta-subunit). Conversely, targeting oncogenesis with a longer fragment of the human alpha-subunit gene (which is activated earlier in development) resulted in the immortalization of a progenitor cell that is more primitive, expressing only the alpha-subunit gene. Interestingly, this transgene also immortalized cells of the thyrotrope lineage that express both alpha- and beta-subunits of thyroid-stimulating hormone and the transcription factor GHF-1 (Pit-1). Thus, targeted tumorigenesis immortalizes mammalian cells at specific stages of differentiation and allows the production of a series of cultured cell lines representing sequential stages of differentiation in a given cell lineage.

Animals↗
PubMed1988

Glucagon gene regulatory region directs oncoprotein expression to neurons and pancreatic alpha cells.

The regulatory region of the rat preproglucagon gene targets expression of the SV40 large T oncoprotein to two cell types in transgenic mice, the pancreatic alpha cells and a set of neurons localized in the hindbrain, both of which normally produce preproglucagon. Additional neurons in the forebrain and midbrain stain for T antigen but do not express the endogenous glucagon gene. Synthesis of T antigen in endocrine alpha cells results in the heritable development of pancreatic glucagonomas. In brains of transgenic mice from three independent lineages, expression of the hybrid gene begins at embryonic day 12 in neuroblasts of the hindbrain, where it continues throughout adult life, most notably in the medulla. Remarkably, oncoprotein expression in both proliferating neuroblasts and mature neurons has no apparent consequences, either phenotypic or tumorigenic. Expression of the hybrid glucagon gene in both neurons and islet cells supports a possible interrelationship between these cell types.

Animals↗
PubMed1990

Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms.

Two cell lines have been established from insulinomas obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice. These cell lines, designated MIN6 and MIN7, produce insulin and T antigen and have morphological characteristics of pancreatic beta cells. MIN6 cells exhibit glucose-inducible insulin secretion comparable with cultured normal mouse islet cells, whereas MIN7 cells do not. Both cell lines produce liver-type glucose transporter (GT) mRNA at high level. Brain-type GT mRNA is also present at considerable level in MIN7 cells, but is barely detectable in MIN6 cells, suggesting that exclusive expression of the liver-type GT is related to glucose-inducible insulin secretion. MIN6 cells do not express either major histocompatibility (MHC) class I or class II antigens on the cell surface. However, treatment with interferon-gamma induces high levels of MHC class I antigens, and a combination of interferon-gamma and tumor necrosis factor-alpha induces a MHC class II antigen on the cell surface. These results emphasize that the MIN6 cell line retains physiological characteristics of normal beta cells. The MIN6 cell line will be especially useful to analyze the molecular mechanisms by which beta cells regulate insulin secretion in response to extracellular glucose concentrations. We discuss a possible role of GT isoforms in glucose sensing by beta cells.

Animals↗
PubMed1998

Enhancement of murine cardiac chronotropy by the molecular transfer of the human beta2 adrenergic receptor cDNA.

Cardiac pacemaking offers a unique opportunity for direct gene transfer into the heart. An experimental system was developed to assay the effects of transferring the human beta2 adrenergic receptor (beta2AR) under in vitro, ex vivo, and finally in vivo conditions. Constructs encoding either beta2AR or LacZ were used in chronotropy studies with isolated myocytes, and transplanted as well as endogenous murine hearts. Murine embryonic cardiac myocytes were transiently transfected with plasmid constructs. The total percentage of myocytes spontaneously contracting was greater in beta2AR transfected cells, as compared with control cells (67 vs. 42+/-5%). In addition, the percentage of myocytes with chronotropic rates > 60 beats per minute (bpm) was higher in the beta2AR population, as compared with control cells (37 vs. 15+/-5%). The average contractile rate was greater in the beta2AR transfected myocytes at baseline (71+/-14 vs. 50+/-10 bpm; P < 0.001) as well as with the addition of 10(-)3 M isoproterenol (98+/-26 vs. 75+/-18 bpm; P < 0.05). Based on these results, a murine neonatal cardiac transplantation model was used to study the ex vivo effects of targeted expression of beta2AR. The constructs were transfected into the right atrium of transplanted hearts. Injection of the beta2AR construct increased the heart rate by approximately 40% (224+/-37 vs. 161+/-42 bpm; P < 0.005). Finally, the constructs were tested in vivo with injection into the right atrium of the endogenous heart. These results were similar to the ex vivo data with injection of the beta2AR constructs increasing the endogenous heart rates by approximately 40%, as compared with control injected hearts (550+/-42 vs. 390+/-37 bpm; P < 0.05). These studies demonstrate that local targeting of gene expression may be a feasible modality to regulate the cardiac pacemaking activity.

Animals↗
PubMed2005

Definition of an immunogenic region within the ovarian tumor antigen stratum corneum chymotryptic enzyme.

PURPOSE: The serine protease stratum corneum chymotryptic enzyme (SCCE) is overexpressed by ovarian tumor cells, but is not expressed by normal tissues, suggesting that SCCE may be an attractive target for immunotherapy. In this study, we tested the hypothesis that dendritic cells loaded with SCCE peptides will induce ovarian tumor antigen-specific CD8+ CTL responses and antigen-specific CD4+ helper T cell responses. EXPERIMENTAL DESIGN: Computer algorithms were used to identify candidate HLA-A2.1-restricted CD8+ CTL epitopes and HLA-DR-binding CD4+ helper T cell epitopes within SCCE. CD8+ CTL stimulated with peptide-loaded dendritic cells were tested against targets expressing endogenous SCCE, including HLA-A2.1-matched ovarian tumor cells. Dendritic cells were also loaded with an extended SCCE peptide, SCCE 110-139, which encompassed a defined CD8+ CTL epitope and multiple candidate CD4+ T helper cell epitopes. RESULTS: CD8+ CTL specific for SCCE 123-131 lysed autologous macrophages infected with an SCCE-expressing recombinant adenovirus, and also lysed HLA-A2.1-matched, SCCE-expressing ovarian tumor cells. Dendritic cells loaded with SCCE 5-13 peptide stimulated an HLA-A2.1-restricted CD8+ CTL response, but with a reduced level of lysis against ovarian tumor cells. Dendritic cells loaded with SCCE 110-139 induced antigen-specific CD4+ T cell and CD8+ T cell responses. Although SCCE 110-139-loaded dendritic cells processed and presented the 123-131 epitope, the dominant CD8+ CTL response was directed against alternative epitopes within SCCE 110-139. CONCLUSIONS: The 110-139 region of SCCE incorporates multiple CD8+ CTL and CD4+ helper T cell epitopes, and represents an attractive target antigen for immunotherapy of ovarian cancer.

Amino Acid Sequence↗
PubMed2003

Ikaros isoforms in human pituitary tumors: distinct localization, histone acetylation, and activation of the 5' fibroblast growth factor receptor-4 promoter.

Targeted expression of a human pituitary tumor derived-fibroblast growth factor receptor-4 (FGFR4) recapitulates pituitary tumorigenesis. We have shown that FGFR4 is a target for Ikaros, a zinc finger-containing transcription factor that localizes to heterochromatin regions and participates in higher order chromatin complexes and control of gene expression. We report here the expression of Ikaros and functional differences between its alternatively spliced variants in human pituitary tumors. Ik1 expression was detected in human pituitary tumors and we also identified a truncated isoform consistent with the non-DNA-binding Ik6 isoform in a subset of adenomas by reverse transcriptase-polymerase chain reaction, sequencing, and Western immunoblotting. Transfection of Ik6 in GH4 pituitary cells resulted in predominantly cytoplasmic expression as compared to Ik1, which resulted in exclusively nuclear expression as determined by immunofluorescence and immunoblotting of fractionated protein. Immunohistochemistry of primary human pituitary adenomas localized Ikaros expression to the nuclear compartment but also in the cytoplasm, the latter consistent with Ik6. Expression of Ikaros and truncated non-DNA-binding isoforms was also suggested by electromobility shift assays using nuclear proteins from primary human pituitary adenomas. Ik6 resulted in reversal of the effects of Ik1 on wild-type 5' FGFR4 promoter activity, histone acetylation, and regulation of the endogenous gene. We conclude that dominant-negative Ik6 isoforms with their distinct localization and effects on Ik1 action may contribute to the altered expression of FGFR4 and possibly other target genes in human pituitary tumors.

Acetylation↗
PubMed2006

Suppression of tumor growth by intratumoral injection of short hairpin RNA-expressing plasmid DNA targeting beta-catenin or hypoxia-inducible factor 1alpha.

To inhibit the growth of murine melanoma B16 cells in mice, we downregulated the gene expression of beta-catenin and hypoxia-inducible factor 1alpha (HIF1alpha) in the tumor cells by delivering short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) targeting one of these genes. Transfection of any of the shRNA-expressing pDNAs to B16 cells resulted in the reduction of the corresponding mRNA, which was associated with a reduced number of viable cells. A flow cytometric analysis of annexin V labeling assay was also performed to count the number of apoptotic cells. A flow cytometric analysis showed that the suppression of the expression of beta-catenin or HIF1alpha in B16 cells increased the number of apoptotic cells. An intratumoral injection of pshbeta-catenin (shRNA-expressing pDNA targeting beta-catenin) or pshHIF1alpha (shRNA-expressing pDNA targeting HIF1alpha) followed by electroporation greatly suppressed the expression of the corresponding target mRNA in the intradermal tumor tissue. The growth of the intradermal tumor was significantly (P<0.05) suppressed by the treatment. In conclusion, tumor growth was successfully inhibited by the intratumoral delivery of pshbeta-catenin or pshHIF1alpha.

Animals↗
PubMed2005

[New strategies to interfere with radiation response: "biomodulation" of radiation therapy].

The development of several new anti cancer agents has been made possible because of recent significant achievements in our global understanding of cancer biology. These new "targeted" agents selectively inhibit targets necessary for tumor cell growth and viability with little toxicity to normal cells compared to conventional cytotoxic agents. So far, the efficacy of many of these new promising agents when used alone treatment remains limited, it is likely that the optimal use of these agents could be obtained in combination with conventional agents such as radiation therapy. The potential benefit of these targeted therapies combined with irradiation seems important. They might offer the advantage of increasing the tumor response to radiation with no or little increase in normal tissue damage. Therefore, these new types of chemo-radiation approaches might respect the normal tissue versus tumor cell "therapeutic ratio". These approaches can be sub divided in three sub groups: 1) Therapeutics targeting selectively one tumor related biochemical activity such as EGFR inhibitors. These approaches are efficient but one mutation of the target might render them inefficient. 2) Therapeutics directed against a widely expressed target. This is the case for anti Insulin Growth Factor-1 (IGF1R) interventions: IGF1R inhibition seems to specifically alter tumor cell viability with a minimal effect on normal cells viability. 3) Strategies which are not targeted against the tumor but the microenvironment, especially angiogenesis. This type of approaches seems to be applicable independently of tumor intrinsic biologic related factors.

Angiogenesis Inhibitors↗
PubMed2026

Antibody-drug conjugates in selected solid tumours: a position statement update based on findings from the third workshop held by the ETOP IBCSG Partners Foundation.

The European Thoracic Oncology Platform (ETOP) International Breast Cancer Study Group (IBCSG) Partners Foundation initiated a series of workshops for experts to review current evidence and offer recommendations to guide future antibody-drug conjugate (ADC) research. Here, we summarise key findings from the third workshop, which included experts in various solid tumours, basic/translational research scientists and pharmaceutical industry representatives. Recent positive phase III trial data have further incorporated ADCs into the standard of care [e.g. lung: sacituzumab tirumotecan; breast: trastuzumab deruxtecan (T-DXd), sacituzumab govitecan, datopotamab deruxtecan; muscle-invasive bladder cancer: enfortumab vedotin; ovarian cancer: mirvetuximab soravastine; cervical cancer: tisotumab vedotin]. Thus, research priorities must be tailored according to tumour type, potentially focussing initially on settings where ADCs could replace chemotherapy. Many phase III ADC trials have been initiated based on positive phase I data and although these trials are larger than those conducted historically, prespecified criteria (e.g. patient numbers and magnitude of efficacy) should be met to justify proceeding directly to phase III. Importantly, although several ADCs have been successfully developed without mandatory biomarker selection, biomarker-driven ADC development enables rational patient selection, as illustrated by multiple ADCs (e.g. T-DXd, mirvetuximab soravtansine and telisotuzumab vedotin). The identification, development and validation of predictive biomarkers are therefore essential, particularly given several critical nuances, including the algorithms used to assess biomarker status and the type of specimen analysed, all of which may be influenced by temporal and spatial heterogeneity. Additional ADC research priorities include the optimisation of ADC constructs to enhance efficacy/tolerability and the identification of reliable ADC targets, including work to elucidate attributes of already-identified targets. Finally, considering the vast amount of ADC-related data being generated, artificial intelligence could be leveraged to analyse combined datasets and generate composite biomarkers, including tumour histology, optimal target expression thresholds, molecular alterations and activated pathways affecting payload activity and target function, to accelerate research.

Humans↗
PubMed2004

Nuclear export of alpha-catenin: overlap between nuclear export signal sequences and the beta-catenin binding site.

Mutations in APC or in beta-catenin, which are common in colon cancer, lead to constitutive activation of beta-catenin/Tcf-dependent signaling. alpha-Catenin is also found in some colon cancer cell nuclei, and loss of its expression correlates with increased beta-catenin/Tcf transcriptional activity. Moreover, targeted expression of alpha-catenin in the nucleus inhibits beta-catenin/Tcf-dependent transcription. Thus, an understanding of the regulation of alpha-catenin localization could provide insight into the control of beta-catenin signaling. While the beta-catenin/Tcf complex can promote nuclear import of alpha-catenin, the mechanism for its nuclear export is not known. We found that leptomycin B (LMB) inhibited nuclear export of GFP-alpha-catenin in HCT116 colon cancer cells, suggesting that alpha-catenin localization is regulated by CRM-1-dependent nuclear export. We identified two putative nuclear export signals in a domain of alpha-catenin that overlaps with the beta-catenin binding domain. Using a nuclear export assay, we determined that one of these (NES1) is a weak LMB-insensitive NES, whereas the other (NES2) is strong and LMB-sensitive. Mutations in either NES reduced nuclear export of alpha-catenin in HCT116 cells. In addition, mutations in NES1, but not NES2, reduced binding of alpha-catenin to beta-catenin and impaired the ability of alpha-catenin to repress beta-catenin/Tcf-dependent transcription. Therefore, NES1 is required both for repression of beta-catenin signaling and for nuclear export, while NES2 is required only for nuclear export.

Active Transport, Cell Nucleus↗
PubMed2002

Keratinocytes constitutively express the CD95 ligand molecule on the plasma membrane: an in situ immunoelectron microscopy study on ultracryosections of normal human skin.

BACKGROUND: Tissue homeostasis is mainly preserved by cytolytic functions. Cytolytic cells, when expressing the CD95 ligand (Fas-L) molecule on the cell membrane, are able to kill CD95 (Fas)-expressing target cells. Although cultured epidermal keratinocytes (KC) have been shown to express Fas-L, and normal skin has been shown to bear Fas-L mRNA, efforts so far to find possible constitutive Fas-L expression on the cell membrane by resting KC in normal human epidermis (i.e. in a functionally active location) have been inconclusive. OBJECTIVES: The aim of the present study was therefore to show the constitutive expression of Fas-L on the plasma membrane of KC. METHODS: Gold immunoelectronmicroscopy, a highly specific and sensitive immunodetection system, was performed in situ on skin sections obtained by ultracryomicrotomy, without previous embedding (i.e. in conditions strictly similar to the in vivo situation). RESULTS: Relatively few (51.55 +/- 28.61), 10-nm colloidal gold particles were observed at the cell surface of KC in the basal layer of the epidermis and an even smaller (P < 0.005) number of gold granules was detected in the KC of the spinous layer. CONCLUSIONS: Although scanty, the constitutive Fas-L expressed on the surface of KC can bind Fas expressed by possible occasional inflammatory cells entering the epidermis, and kill them, so preventing inflammation. Fas-L-expressing KC could moreover induce apoptosis of epidermal cells bearing viral or neoplastic antigens. Thus, the expression of Fas-L by KC may contribute to the preservation of epidermal homeostasis in vivo.

Apoptosis↗
PubMed2002

Bone morphogenetic protein signals are required for cartilage formation and differently regulate joint development during skeletogenesis.

The bone morphogenetic protein (BMP) family consists of a large number of members and has diverse biological activities during development. Various tissues express pleural BMP family members, which seem to cooperatively regulate developmental events. Here, multiple BMP signals were inactivated in chondrocytes to clarify the function of BMPs during skeletogenesis. To obtain tissue-specific inactivation, Noggin gene (Nog) was overexpressed in cartilage under the control of a2(XI) collagen gene (Collla2) promoter/enhancer sequences. The resultant transgenic mice lacked most of their cartilaginous components, suggesting that cartilage does not develop without BMP signals. These effects seem to be mediated through down-regulation of Sox9 expression. Conversely, specific BMP signals were activated in the skeleton by targeted expression of Bmp4 in cartilage and the resultant phenotype was compared with that of transgenic mice expressing growth and differentiation factor-5 (GDF-5), another BMP family member. Overactivity of Bmp4 in the skeleton caused an increase of cartilage production and enhanced chondrocyte differentiation, as GDF5 expression did, but it did not disturb joint formation as GDF5 did. During skeletogenesis, unique roles of each BMP may reside in the regulation of joint development. Together with the common effect on the cartilage overproduction by Bmp4 and GDF5 overactivation, loss of cartilage by inactivation of multiple BMPs in Noggin transgenic mice indicates that signals for cartilage production are reinforced by multiple BMPs exclusively. These conclusions may account for the reason why multiple BMPs are coexpressed in cartilage.

Animals↗
PubMed1998

Severe follicular hyperplasia and spontaneous papilloma formation in transgenic mice expressing the neu oncogene under the control of the bovine keratin 5 promoter.

Transgenic mice were developed to explore the role of the erbB2 during epithelial homeostasis and tumorigenesis, through targeted expression of the neu oncogene (neu*). Expression of a neu* cDNA was targeted to the basal layer of skin epidermis as well as other epithelial tissues of transgenic mice via the bovine keratin 5 promoter. Two transgenic founders were obtained that were morphologically distinguishable from non-transgenic littermates by their visibly thickened skin and patchy hair growth by day 3 after birth. The presence of the transgene was confirmed by polymerase chain reaction analysis of tail DNA and immunofluorescence analysis of neu* protein in skin sections. Histological evaluation revealed significant hyperplasia of the follicular and interfollicular epidermis, the abnormal presence of horny material in the dermis and hypodermis, and a dramatic increase in epidermal proliferation. Many areas of the dermis involving this abnormal epithelial proliferation exhibited a squamous cell carcinoma-like appearance. In addition, there was unusual proliferation of the sebaceous glands. One founder died at day 14 and the other at day 20. The latter founder had two papillomas at the time of death. Additional phenotypic changes resulting from the expression of neu* in other tissues included hyperkeratosis in the forestomach and esophagus. In addition, there was a lack of distinction of the cortical-medullary boundaries and an increased rate of cell death in lymphocytes in the thymus. The phenotypic changes in these other tissues correlated with transgene expression. The data suggest that erbB2 signaling has an important role in epidermal proliferation. In addition, the data provide strong support for a role for erbB2 signaling during epidermal carcinogenesis in mouse skin.

Animals↗
PubMed1998

Interleukin-12-activated natural killer cells recognize B7 costimulatory molecules on tumor cells and autologous dendritic cells.

Activation of natural killer (NK) cells in the presence of interleukin-12 (IL-12) augments the capacity of these effector cells to recognize B7-1- and B7-2-expressing target cells. These effector cells also efficiently lyse autologous B7-positive progenitor or organ-derived dendritic cells, suggesting a physiologic regulatory pathway between IL-12, NK cells, and B7-expressing antigen-presenting cells. Although IL-12-activated NK cells secreted higher levels of interferon-gamma, this cytokine did not play a role in synergistic effects of IL-12 and B7 on NK activation. The B7-counterreceptor was found to be selectively upregulated on IL-2/IL-12 as compared with IL-2-activated NK cells. CD28 is functionally involved in the recognition of B7 on target cells since IL-2/IL-12-activated NK cells derived from CD28 knockout mice were strongly reduced in their capacity to lyse syngeneic B7-positive tumor cells as well as antigen-presenting cells. However, recognition of B7 on allogeneic targets did not require the expression of CD28 on the IL-2/IL-12-activated NK cells. Hence, IL-12 triggers the expression of both CD28-dependent and CD28-independent mechanisms that allow NK cells to eliminate B7-positive target cells including autologous dendritic cells.

Animals↗
PubMedDate not supplied

p53 down-regulates human bradykinin B1 receptor gene expression.

The tumor suppressor, p53, has been shown to transcriptionally activate or silence a number of target genes. As an activator, p53 relies on its specific consensus sequence within the promoter. It is not clear whether p53 requires a specific DNA binding site in its action as a gene repressor. This report demonstrates that the human BKB1R gene is a p53 target. Expression of p53 in transiently transfected SV40-transformed IMR90 cells strongly suppressed luciferase reporter activity driven by a 1.8 kb BKB1R promoter as well as its minigene. These down-regulations were p53 dose-dependent. p53 reduced both basal and induced promoter activities of the minigene. Expression of p53 abolished the inducibility of the minigene. Induction of endogenous p53 expression by etoposide also inhibited promoter activity and minigene inducibility. Replacing the region containing both the putative p53 binding site and the TATA-box with a basal adenovirus promoter in the 1.8 kb promoter construct did not prevent p53 from inhibiting BKB1R promoter activity. Thus suppression by p53 is not mediated by competition with the TATA-binding protein and is not through interaction with the putative p53-binding site. p53 also does not appear to suppress BKB1R gene expression through interaction with c-Jun which functions in the inducibility of this gene [Yang et al., 2001].

Binding Sites↗
PubMedDate not supplied

Primary polyclonal human T lymphocytes targeted to carcino-embryonic antigens and neural cell adhesion molecule tumor antigens by CD3zeta-based chimeric immune receptors.

Antigen-specific T lymphocytes are attractive as potential anticancer agents. The generation of large numbers of antigen-specific T cells is possible through the use of gene therapy to express targeting receptors on the T lymphocyte. Activated T lymphocytes were transduced to express carcino-embryonic antigen or neural cell adhesion molecule targeted CD3zeta chimeric immune receptors. The chimeric receptors were expressed as homodimers and also as heterodimers with the native CD3zeta. T lymphocyte populations were expanded in the absence of selection for the modified cells and were shown to produce cytokines when cultured in the presence of immobilized purified protein antigen. These lymphocytes also responded by cytokine production and cytolytic activity when challenged with tumor-cell lines expressing the antigen recognized by the chimeric immune receptor. The cytolytic activity appears to be largely perforin mediated. Furthermore, soluble carcino-embryonic antigen did not interfere with the functional activity of the carcino-embryonic antigen-targeted lymphocytes. Long-term (5-day) stimulation of the modified lymphocytes by protein antigen resulted in reduced viability similar to that induced by anti-CD3 antibodies alone. Viability was improved by a costimulatory signal indicating that such signals may be vital in the maintenance of long-term functional activity of receptor modified T lymphocytes.

Antigens, Neoplasm↗