Beta-gal Expression Research Articles

Abstract 1345: p21 (WAF1/CIP1) drives a secretory phenotype in senescent fibroblasts and promotes bystander cancer cell growth in the tumor microenvironment

Abstract Cancer associated fibroblasts (CAF) are major stromal cells in the tumor microenvironment (TME) and support cancer cell growth. Chemotherapy can induce CAF senescence in which p21 (the cyclin-dependent kinase inhibitor) permanently arrests the cell cycle. Cellular senescence and its associated secretory phenotype (SASP) can promote cancer progression and lead to drug resistance. We explored molecular mechanisms of p21 in the cellular communication between cancer and senescent fibroblasts. We induced fibroblast cell senescence by treatment with etoposide. Cellular senescence was confirmed by cytokine profiling, beta-gal staining and high p21 expression. To evaluate the role of p21 in the SASP of senescent fibroblasts, we transiently knocked down p21 expression in senescent fibroblast cells using siRNA. SA-beta-gal staining showed a strong beta-gal expression around nuclei in the senescent fibroblast cells with the knockdown of p21, which is the same as it was in the cells with siRNA control. Further cytokine profiling showed two panels of SASP upon p21 knockdown in senescent cells: 1) decrease of cytokines such as CCL2 and IL8 and 2) increase of cytokines such TNF-alpha correlated to the knockdown of p21 in the senescent cells. These results suggest that p21 drives a SASP in senescent fibroblast cells, in addition to its function in cell cycle arrest. We examined the effect of the p21-driven SASP in senescent fibroblasts on bystander cancer cell growth in the TME. Our co-culture system showed knockdown of p21 in senescent fibroblasts suppressed bystander cancer cell growth. We found that knockdown of TNF partially blocked the antiproliferation effect of p21-knockdown on bystander cancer cells. Our results suggest that senescent fibroblasts support bystander cancer cell growth via a p21-driven SASP in the TME and provides a new strategy for targeting p21-driven SASP in the TME for cancer therapy. Citation Format: Shengliang Zhang, Kelsey E. Huntington, Lanlan Zhou, Wafik S. El-Deiry. p21 (WAF1/CIP1) drives a secretory phenotype in senescent fibroblasts and promotes bystander cancer cell growth in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1345.

Abstract 12245: Surface Lin28a Expression Indicative of Cellular Stress Directed Towards Senescence

Introduction: Declining cellular functional capacity resulting from stress or aging is a primary contributor to impairment of myocardial performance. Molecular pathway regulation of biological processes in cardiac interstitial cells (CICs) is pivotal in stress and aging responses. Altered localization of the RNA binding protein Lin28A has been reported in response to environmental stress, but the role of Lin28A in response to stress in CICs has not been explored. Hypothesis: Lin28A redistribution is indicative of stress response in CIC associated with aging and senescence. Methods and Results: Localization of Lin28A was assessed by multiple experimental analyses and treatment conditions and correlated to oxidative stress, senescence, and ploidy in adult murine CICs. Surface Lin28A expression is present on 5% of fresh CICs and maintained through passage 2, increasing to 21% in hyperoxic conditions but lowered to 14% in physiologic normoxia. Surface Lin28A is coincident with elevated Beta-galactosidase (Beta-gal) expression in CICs expanded in hyperoxia, and also increases with polyploidization and binucleation of CICs regardless of oxygen culture. Transcriptional profiling of CICs using single cell RNASeq reveals upregulation of pathways associated with oxidative stress in CICs exhibiting surface Lin28A. Induction of surface Lin28A by oxidative stress is blunted by treatment of cells with the antioxidant Trolox in a dose-dependent manner, with 300uM Trolox exposure maintaining characteristics of freshly isolated CICs possessing low expression of surface Lin28A and Beta-gal with predominantly diploid content. Conclusion: Surface Lin28A is a marker of environmental oxidative stress in CICs and antioxidant treatment antagonizes this phenotype. The biological significance of Lin28 surface expression and consequences for myocardial responses may provide important insights regarding mitigation of cardiac stress and aging.

Disruption of a Spermatogenic Cell-Specific Mouse Enolase 4 (Eno4) Gene Causes Sperm Structural Defects and Male Infertility1

Sperm utilize glycolysis to generate ATP required for motility, and several spermatogenic cell-specific glycolytic isozymes are associated with the fibrous sheath (FS) in the principal piece of the sperm flagellum. We used proteomics and molecular biology approaches to confirm earlier reports that a novel enolase is present in mouse sperm. We then found that a pan-enolase antibody, but not antibodies to ENO2 and ENO3, recognized a protein in the principal piece of the mouse sperm flagellum. Database analyses identified two previously uncharacterized enolase family-like candidate genes, 64306537H0Rik and Gm5506. Northern analysis indicated that 64306537H0Rik (renamed Eno4) was transcribed in testes of mice by Postnatal Day 12. To determine the role of ENO4, we generated mice using embryonic stem cells in which an Eno4 allele was disrupted by a gene trap containing a beta galactosidase (beta-gal) reporter (Eno4(+/Gt)). Expression of beta-gal occurred in the testis, and male mice homozygous for the gene trap allele (Eno4(Gt/Gt)) were infertile. Epididymal sperm numbers were 2-fold lower and sperm motility was reduced substantially in Eno4(Gt/Gt) mice compared to wild-type mice. Sperm from Eno4(Gt/Gt) mice had a coiled flagellum and a disorganized FS. The Gm5506 gene encodes a protein identical to ENO1 and also is transcribed at a low level in testis. We conclude that ENO4 is required for normal assembly of the FS and provides most of the enolase activity in sperm and that Eno1 and/or Gm5506 may encode a minor portion of the enolase activity in sperm.

Epidermal γδ T cells sense precancerous cellular dysregulation and initiate immune responses

Hyperplasia associated with a loss of tissue homeostasis can induce DNA replication stress, leading to precancerous dysregulation. Epidermal gammadelta T cells reside in the primary barrier that protects against diverse environmental insults; however, the functions of these T cells in tissue surveillance are not completely understood. In mice with inducible Notch1 inactivation in keratinocytes that causes epidermal hyperplasia, epidermal gammadelta T cells sensed stressed keratinocytes and migrated into the cutaneous draining lymph nodes. Simultaneous induction of beta-galactosidase (beta-Gal) as a putative antigen expressed in the process of precancerous dysregulation and Notch1 ablation in the epidermis resulted in elevated beta-Gal-specific IgG2a production. Epidermal gammadelta T cells were found to have the capacity to express chemokine (C-C motif) receptor 7 and migrate into the lymph nodes. Cutaneous draining lymph node cells in Notch1-inactivated mice expressed high levels of IFN-gamma upon anti-CD3 plus anti-CD28 stimulation. Furthermore, induced expression of beta-Gal in mice that lacked epidermal gammadelta T cells failed to induce anti-beta-Gal IgG. These results suggest that epidermal gammadelta T cells play an essential role in the initiation process of epidermal antigen-specific humoral immune responses and demonstrate the importance of epidermal gammadelta T cells in sensing precancerous dysregulation and activating adaptive immunity.

Strong Muscle-Specific Regulatory Cassettes Based on Multiple Copies of the Human Slow Troponin I Gene Upstream Enhancer

High-level tissue-specific expression of recombinant proteins in muscle is an important issue for several therapeutic applications. To achieve this goal, we generated several constructs containing one to five copies of the upstream enhancer (USE) of 160-bp of the human slow troponin I gene, linked to that gene's minimal promoter. We also tested constructs made with one to four copies of a 100-bp deletion of USE (DeltaUSE) reported to drive pan-muscle-specific expression in transgenic mice. These constructs were evaluated by measuring the activity of the reporter gene beta-galactosidase (beta-gal). In cell culture, these multimerized enhancers retained tissue specificity and their transcriptional strength increased with the number of enhancer copies. In myotube cultures (which still contain nondifferentiated cells), constructs containing four and five USE copies were stronger than the cytomegalovirus (CMV) early enhancer/promoter and slightly weaker than the hybrid CMV enhancer/beta-actin (CB) promoter. Those containing three USE, or four DeltaUSE copies were similar in strength to CMV. After electrotransfer of plasmid DNA into the mouse tibialis anterior muscle, the strengths of the two constructs (USEx3 and DeltaUSEx3) were tested; as measured by beta-gal activity in the total muscle lysate and by the number of transduced fibers, they were similar to CMV and CB. Muscle fiber typing, after electrotransfer of the soleus muscle, showed that DeltaUSEx3 and USEx3 were active in slow and fast fibers. The tissue specificity of these two constructs was also evaluated by hydrodynamic plasmid injection through the tail vein. Although significant beta-gal expression was measured in the liver when CMV was tested, no expression above background level was detected with USEx3 and DeltaUSEx3. The strength, muscle specificity, and small size of these transcriptional elements render them very attractive for gene therapy applications.

Expression of Runx2 transcription factor in non‐skeletal tissues, sperm and brain

Runx2 is a master transcription factor for chondrocyte and osteoblast differentiation and bone formation. However expression of Runx2 (by RT-PCR), has been reported in non-skeletal tissues such as breast, T cells and testis. To better define Runx2 activity in non-skeletal tissues, we examined transgenic (Tg) mice expressing LacZ gene under control of 3.0 kb (3 kb Tg) or 1.0 kb (1 kb Tg) of the Runx2 distal (P1) promoter, Runx2 LacZ knock-in (Runx2(+/LacZ)) and Runx2/P1 LacZ knock-in (Runx2/P1(+/LacZ)). In the Runx2 3 kb Tg mouse, beta-galactosidase (beta-gal) expression appeared in various non-skeletal tissues including testis, skin, adrenal gland and brain. beta-gal expression from both 3 kb and 1 kb Tg, reflecting activity of the Runx2 promoter, was readily detectable in seminiferous tubules of the testis and the epididymis. At the single cell level, beta-gal was detected in spermatids and mature sperms not in sertoli or Leydig cells. We also detected a positive signal from the Runx2(+/LacZ) and Runx2/P1(+/LacZ) mice. Indeed, Runx2 expression was observed in isolated mature sperms, which was confirmed by RT-PCR and Western blot analysis. Runx2, however, was not related to sex determination and sperm motility. Runx2 mediated beta-gal activity is also found robustly in the hippocampus and frontal lobe of the brain in Runx2(+/LacZ). Collectively, these results indicate that Runx2 is expressed in several non-skeletal tissues particularly sperms of testis and hippocampus of brain. It suggests that Runx2 may play an important role in male reproductive organ testis and brain.

BDNF Promoter-Mediated -Galactosidase Expression in the Olfactory Epithelium and Bulb

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in the generation and differentiation of new olfactory sensory neurons (OSNs) and in the regulation of branching of OSN axons in their target glomeruli. However, previous reports of BDNF mRNA and protein expression in olfactory epithelium and olfactory bulb (OB) have been inconsistent, raising questions on the proposed roles for BDNF. Here, we report on β-galactosidase (β-gal) expression in adult gene-targeted mice where the BDNF promoter drives expression of the Escherichia coli lacZ gene (BDNFlacZneo mice). We find that β-gal is expressed in a small subset of OSNs with axons that reach the olfactory nerve layers throughout the OB. In the OB, we find expression of β-gal in γ-aminobutyric acidergic but not dopaminergic periglomerular cells and external tufted cells and in interneurons located in the mitral cell layer. Our results are inconsistent with the regulation of generation and differentiation of new OSNs elicited by the release of BDNF from horizontal basal cells. The results are consistent with a role for BDNF in competitive branching of OSN axons within the glomeruli of the OB.

Targeted Cornea Limbal Stem/Progenitor Cell Transfection in an Organ Culture Model

To optimize a nonviral gene transfection system targeting the corneal limbal stem/progenitor cells. A plasmid containing LacZ gene coding for beta-galactosidase (beta-gal) was transfected into human corneal epithelial cells (HCECs) and multilineage progenitor cells (MLPCs) with different transfection reagents, to determine the optimal transfection reagent. In an ex vivo study, the bovine corneal epithelium and limbal stem/progenitor cells were transfected with a microinjection system with a 36-gauge needle that delivered plasmid/transfection reagent (Lipofectamine 2000; Invitrogen, Carlsbad, CA) complexes. The transfected corneoscleral discs were cultured in an air-interface culture system. The expression of beta-gal was determined with an X-gal staining assay, and images were acquired with light microscopy and transmission electron microscopy. The expression of cytokeratin K5/14 and K3/K12 in corneal and limbal epithelium was determined by immunohistochemistry. The highest percentages of beta-gal expression in HCECs and MLPCs were achieved when the transfection reagent Lipofectamine 2000 was used. Corneal epithelial and limbal basal cells were successfully transfected with the reporter gene by targeted microinjection of plasmid/liposomal complexes. The location of the bovine limbal stem/progenitor cells was confirmed by positive K5/K14 labeling and negative K3/12 labeling. Targeted microinjection of plasmid/liposomal complexes resulted in limbal stem/progenitor cell transfection. This technique has potential for the short-term treatment of corneal diseases.

A unique cell population in the mouse olfactory bulb displays nuclear β‐catenin signaling during development and olfactory sensory neuron regeneration

Olfactory sensory neurons (OSNs) in the nose form precise connections with neurons in the brain. However, mechanisms that account for the formation of such precise neuronal connections are incompletely understood. Recent studies implicate the function of Wnt growth factors in the formation of neuronal connections. To assess the role of Wnt signaling in the olfactory system, we examined the expression of beta-galactosidase (beta-gal) in the TOPGAL mouse, a transgenic strain in which beta-gal expression reports the activation of the canonical Wnt signaling pathway. In the olfactory epithelium, no beta-gal expression was observed at any developmental stages. In the olfactory bulb (OB), beta-gal expression was observed in a population of cells located at the interface of the olfactory nerve layer and the glomerular layer. The beta-gal expression was developmentally regulated with the peak expression occurring at late embryonic and early postnatal stages and a greatly reduced expression in adulthood. Further, forced OSN regeneration and subsequent reinnervation of the OB led to a reactivation of beta-gal expression in mature animals. The temporal coincidence between the peak of beta-gal expression and formation of OSN connections, together with the spatial localization of these cells, suggests a potential role of these cells and canonical Wnt signaling in the formation of OSN connections in the OB during development and regeneration.

Immunization Against the Transgene but not the TetON Switch Reduces Expression From Gutless Adenoviral Vectors in the Brain

Immune responses against vectors or encoded transgenes can impose limitations on gene therapy. We demonstrated that tetracycline-regulated high-capacity adenoviral vectors (HC-Ads) sustain regulated transgene expression in the brain even in the presence of systemic pre-existing immune responses against adenoviruses. In this study we assessed whether systemic pre-existing immune responses against the transgene products, i.e., beta-Gal or the tetracycline-dependent (TetON) regulatory transcription factors (rtTA2(S)M2 and the tTS(Kid)), affect transgene expression levels and the safety profile of HC-Ads in the brain. We pre-immunized mice with plasmids encoding the TetON switch expressing rtTA2(S)M2 and the tTS(Kid) or beta-Gal. HC-Ads expressing beta-Gal under the control of the TetON switch were then injected into the striatum. We assessed levels and distribution of beta-Gal expression, and evaluated local inflammation and neuropathological changes. We found that systemic immunity against beta-Gal, but not against the TetON switch, led to inflammation and reduction of transgene expression in the striatum. Therefore, the regulatory TetON switch appears to be safe to use, and capable of sustaining transgene expression in the brain even in the presence of an immune response against its components. Systemic immunity against the transgene had the effect of curtailing its expression, thereby affecting the efficacy and safety of gene delivery to the brain. This factor should be considered when developing gene therapies for neurological use.

A mesenchymal perspective of Müllerian duct differentiation and regression in Amhr2-lacZ mice.

The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Müllerian ducts regress, preventing the formation of female organs. We introduced the bacterial lacZ gene, encoding beta-galactosidase (beta-gal), into the AMHR-II locus (Amhr2) by gene targeting in mouse embryonic stem (ES) cells to mark Müllerian duct differentiation and regression. We show that Amhr2-lacZ heterozygotes express beta-gal activity in an Amhr2-specific pattern. In the gonads, beta-gal activity was detected in Sertoli cells of the testes from 2 weeks after birth, and fetal ovaries and granulosa cells of the adult ovary. beta-gal activity was first detected in the rostral mesenchyme of the Müllerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Müllerian ducts with higher levels initially found in males. In females, beta-gal activity was restricted to one side of the ductal mesoepithelium, whereas in males beta-gal expression encircled the duct. beta-gal activity was also detected in the coelomic epithelium at 13.5 and 14.5 dpc. In male embryos, mesenchymal beta-gal activity permitted the visualization of the temporal and spatial pattern of Müllerian duct regression. This pattern was similar to that observed using a Müllerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Müllerian duct mesoepithelium and Amhr2-expressing mesenchyme.

B‐Myb regulates the A2B adenosine receptor in vascular smooth muscle cells

The A(2B) adenosine receptor (A(2B)AR) has been described to control various vascular functions, including inhibition of smooth muscle cell proliferation. Here, we sought to understand the regulation of A(2B)AR gene expression in aortic vascular smooth muscle cells (VSMCs), with a focus on the proliferation phase. Assays with A(2B)AR-beta-gal aortic VSMCs, in which the endogenous A(2B)AR gene promoter drives the expression of prokaryotic beta-galactosidase (beta-gal) instead of the endogenous A(2B)AR gene, show that beta-gal expression is upregulated when the cells are induced to exit from cell cycle arrest. Similarly, the level of A(2B)AR mRNA is upregulated in proliferating primary aortic VSMCs. In search of related mechanisms, it was noted that the A(2B)AR gene promoter contains several putative binding sites for the proliferation-inducing transcription factor, B-Myb. Using a clone of the 5' region upstream of the mouse A(2B)AR gene linked to a reporter gene, B-Myb site deletion mutants were generated. It was determined that B-Myb upregulates the A(2B)AR gene promoter, and specific promoter binding sites were identified as functional. In accordance, B-Myb also elevates endogenous A(2B)AR mRNA and receptor activity, and this activity decreases cell proliferation. Our data are novel in that they show that this proliferation-inhibiting A(2B)AR is itself an inducible receptor regulated by B-Myb.

Non-invasive in vivo optical imaging of the lacZ and luc gene expression in mice

The bacterial lacZ gene encoding for beta-galactosidase (beta-gal) is a common reporter gene used in transgenic mice. Nonetheless, the absence of fluorigenic substrates usable in live animals greatly hampered the non-invasive follow-up of this reporter gene expression. We used far-red fluorescence for imaging beta-Gal expression in live cells in vitro or in vivo. The 9H-(1,3-dichloro-9,9-dimethylacridin- 2-one-7-yl) beta-D-galactopyranoside substrate was used to monitor beta-Gal expression as a reporter of tumor growth, or of the physiological levels of an endogenous gene or of gene transfer in lung. A quantitative evaluation of this method as well as a comparison of its sensitivity with Firefly Luciferase-based bioluminescence was also performed. In vivo measurements showed that 10(3) beta-Gal tumor cells located under the skin were detectable. In deeper organs like lung, as little as 5 ng of beta-Gal or Luciferase enzymes per mg of proteins were measured, confirming that both techniques reached similar sensibilities. Nonetheless, quantitative comparison of beta-Gal levels measured with far-red imaging or with a standardized enzymatic evaluation after killing revealed that the 2D-fluorescent reflectance imaging method is submitted to a color-dependent disparity of the organs and cannot supply quantitative measurements but that a simple correction can be applied.

Integration of active human β-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector

Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150 kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal beta-galactosidase (BGAL) gene of 100 kb. Temporary expression and functional activity of beta-galactosidase (beta-gal) could be detected in human beta-gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of beta-gal was achieved in Gli36 cells infected with rep-plus, but not rep-minus, HSV/AAV hybrid vectors. None of five clones isolated after rep-minus hybrid vector infection showed elevated beta-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed beta-gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep-plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and beta-gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.

Reduced host cell reactivation of oxidative DNA damage in human cells deficient in the mismatch repair gene hMSH2

Germ line mutations in the mismatch repair (MMR) genes hMSH2 and hMLH1 account for approximately 98% of hereditary non-polyposis colorectal cancers. In addition, there is increasing evidence for an involvement of MMR gene expression in the response of cells to UV-induced skin cancer. The link between MMR and skin cancer suggests an involvement of MMR gene expression in the response of skin cells to UV-induced DNA damage. In this report, we have used two reporter gene assays to examine the role of hMSH2 and hMLH1 in the repair of oxidative DNA damage induced by UVA light and DNA damage caused by methylene blue plus visible light (MB+VL). UVA and MB+VL produce 8-hydroxyguanines in DNA that are repaired by base excision repair (BER). AdHCMVlacZ is a replication-deficient recombinant adenovirus that expresses the beta-galactosidase (beta-gal) reporter gene under the control of the human cytomegalovirus (CMV) immediate-early promoter. We show a reduced host cell reactivation for beta-gal expression of UVA-treated and MB+VL-treated AdHCMVlacZ in hMSH2-deficient LoVo human colon adenocarcinoma cells compared to their hMSH2-proficient counterpart SW480 cells, but not in hMLH1-deficient HCT116 human colon adenocarcinoma cells compared to hMLH1-proficient HCT116-chr3 cells. We have also reported previously that enhanced expression of the undamaged AdHCMVlacZ reporter gene is induced by the pre-treatment of cells with lower levels of the DNA-damaging agent and to higher expression levels in transcription-coupled repair (TCR)-deficient compared to TCR-proficient cells. Here we show that pre-treatment of cells with UVA or MB+VL enhanced expression of the undamaged reporter gene to a higher level in LoVo compared to SW480 cells but there was little or no difference in HCT116 compared to HCT116-chr3 cells. These results suggest a substantial involvement of hMSH2 but little or no involvement of hMLH1 in the repair of UVA- and MB+VL-induced oxidative DNA damage by BER.

A method for detecting functional activity related expression in gross brain regions, specific brain nuclei and individual neuronal cell bodies and their projections

We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves detection of neuronal activation by histochemical or immunohistochemical detection of β-galactosidase (βgal), the product of the lacZ gene. Furthermore, the underlying brain state of the FTL mice determines the basal levels of expression of βgal. Here we describe in detail our protocols for detection of FTL expression in these mice and discuss the main variables which need to be considered in the use of these mice for the detection and mapping of functionally activated neurons, circuits and regions in the brain.

Expression and Externalization of Annexin 1 in the Adrenal Gland: Structure and Function of the Adrenal Gland in Annexin 1-Null Mutant Mice

Annexin 1 (ANXA1) is a member of the annexin family of phospholipid- and calcium-binding proteins with a well demonstrated role in early delayed (30 min to 3 h) inhibitory feedback of glucocorticoids in the hypothalamus and pituitary gland. This study used adrenal gland tissue from ANXA1-null transgenic mice, in which a beta-galactosidase (beta-Gal) reporter gene was controlled by the ANXA1 promoter, and wild-type control mice to explore the potential role of ANXA1 in adrenal function. RT-PCR and Western blotting revealed strong expression of ANXA1 mRNA and protein in the adrenal gland. Immunofluorescence labeling of ANXA1 in wild-type and beta-Gal expression in ANXA1-null adrenals localized intense staining in the outer perimeter cell layers. Immunogold electron microscopy identified cytoplasmic and nuclear ANXA1 labeling in outer cortical cells and capsular cells. Exposure of adrenal segments in vitro to dexamethasone (0.1 mum, 3 h) caused an increase in the amount of ANXA1 in the intracellular compartment and attached to the surface of the cells. The N-terminal peptide ANXA1(Ac2-26) inhibited corticosterone release. Corticosterone release was significantly greater from ANXA1-null adrenal cells compared with wild type in response to ACTH (10 pm to 5 nm). In contrast, basal and ACTH-stimulated aldosterone release from ANXA1-null adrenal cells was not different from wild type. Morphometry studies demonstrated that ANXA1 null adrenal glands were smaller than wild-type, and the cortical/medullary area ratio was significantly reduced. These results suggest ANXA1 is a regulator of adrenocortical size and corticosterone secretion.

DNA damage and oncogenic protein expression induced by cadmium

To explore the DNA damage and the expression of oncogenic protein induced by cadmium in vitro (human cells) and in vivo in rats. The colony formation assay and the MTT assay were employed to determine the cytotoxicity of cadmium. The DNA damage and the cell cycle were measured by the comet assay and the flow cytometry, respectively. The western bolt and the X-Gal staining were also used to determine the change of oncogenic protein and the senescent marker. The cadmium inhibited the proliferation of cells and induced DNA damage significantly not only in human cultured cells but also in vivo animal cells. The comet rate increased from 6.1% in the control group to 23.2% in 200 microM cadmium treatment group (P < 0.01). The comet rate increased in all organs of male rats with the increase of dosage, and there were significant difference between treatment the groups and the control group in kidney and ventral prostate (P < 0.05). Furthermore, the cadmium blocked the cell cycle progression. At the same time, the expression of c-myc, c-Jun and beta-Gal were increased by cadmium. The cadmium could induce DNA damage and block the cell cycle, and further cause senescence, and death. However, the cadmium induced the DNA damage and the oncogenic protein expression may be the important factors to cause cancer.

Regulation of the Neurofibromatosis 2 gene promoter expression during embryonic development

Mutations in the Neurofibromatosis 2 (NF2) gene are associated with predisposition to vestibular schwannomas, spinal schwannomas, meningiomas, and ependymomas. Presently, how NF2 is expressed during embryonic development and in the tissues affected by neurofibromatosis type 2 (NF2) has not been well defined. To examine NF2 expression in vivo, we generated transgenic mice carrying a 2.4-kb NF2 promoter driving beta-galactosidase (beta-gal) with a nuclear localization signal. Whole-mount embryo staining revealed that the NF2 promoter directed beta-gal expression as early as embryonic day E5.5. Strong expression was detected at E6.5 in the embryonic ectoderm containing many mitotic cells. beta-gal staining was also found in parts of embryonic endoderm and mesoderm. The beta-gal staining pattern in the embryonic tissues was corroborated by in situ hybridization analysis of endogenous Nf2 RNA expression. Importantly, we observed strong NF2 promoter activity in the developing brain and in sites containing migrating cells including the neural tube closure, branchial arches, dorsal aorta, and paraaortic splanchnopleura. Furthermore, we noted a transient change of NF2 promoter activity during neural crest cell migration. While little beta-gal activity was detected in premigratory neural crest cells at the dorsal ridge region of the neural fold, significant activity was seen in the neural crest cells already migrating away from the dorsal neural tube. In addition, we detected considerable NF2 promoter activity in various NF2-affected tissues such as acoustic ganglion, trigeminal ganglion, spinal ganglia, optic chiasma, the ependymal cell-containing tela choroidea, and the pigmented epithelium of the retina. The NF2 promoter expression pattern during embryogenesis suggests a specific regulation of the NF2 gene during neural crest cell migration and further supports the role of merlin in cell adhesion, motility, and proliferation during development.

High volume hydrodynamic injection of plasmid DNA via the hepatic artery results in a high level of gene expression in rat hepatocellular carcinoma induced by diethylnitrosamine

Hydrodynamic injection of naked plasmid DNA (pDNA) via the tail vein is a safe and effective method of gene transfer to the liver. However, successful gene transfer has yet to be shown for hepatocellular carcinoma (HCC); therefore, we investigated the feasibility and efficacy of hydrodynamic injection via the tail vein and hepatic artery in a diethylnitrosamine (DEN)-induced HCC model in rats. HCC was induced in Sprague-Dawley rats by 100 ppm DEN in drinking water. pCMV-SPORT-beta-galactosidase (beta-gal, 400 microg) was injected (i) via the tail vein in a volume of 0.1 ml/g in 30 s or (ii) via the hepatic artery in a volume of 5 or 10 ml at 1 ml/s, either with or without temporary occlusion of the inferior vena cava (IVC) and portal vein (PV). The liver was harvested 24 h after administration, and beta-gal expression was evaluated with X-gal staining and measurement of enzymatic activity in tissue homogenates. Hydrodynamic injection via the tail vein achieved transgene expression only in non-cancerous tissue (tumor: 0.16 +/- 0.04%, non-tumor: 5.07 +/- 1.66%). Hydrodynamic injection via the hepatic artery was tolerated, but failed to produce efficient transgene expression in tumor and non-tumor cells. On the other hand, concomitant use of temporary IVC/PV occlusion with hydrodynamic injection via the hepatic artery dramatically increased transgene expression in cancer cells, but tumor-selective gene transfer was not achieved with this procedure (tumor: 7.38 +/- 3.66%, non-tumor: 7.77 +/- 1.06%). High-volume hydrodynamic injection of a pDNA solution via the hepatic artery with IVC/PV occlusion achieved a high level of gene expression in a HCC rat model. This gene transfer technique may have potential in clinical gene therapy for HCC.