Sachiko Matsuhashi

Loss of PDCD4 expression in human lung cancer correlates with tumour progression and prognosis

The programmed cell death 4 gene (PDCD4), a newly identified transformation suppressor, was analysed in lung tumour cell lines and primary lung carcinomas. Reduced PDCD4 mRNA expression was observed in two immortalized lung cell lines and 18 cancer cell lines by northern blot analysis. In the survey of primary lung tumours, PDCD4 cDNA was poorly represented in 47 lung tumours compared with normal lung tissue by cDNA microarray analysis and this poor representation was significantly associated with high‐grade (G3) adenocarcinomas (p = 0.012). Immunohistochemical analysis of 124 primary carcinomas comprising all subtypes demonstrated that PDCD4 protein expression was widely lost in tumour samples (83%) and was negatively related to poor prognosis (p = 0.013). The loss of PDCD4 expression correlated with higher grade and disease stage (p = 0.045 and 0.034, respectively), but not tumour size and nodal status. Similarly to the cDNA data, lack of PDCD4 expression was significantly linked to tumour grade in adenocarcinoma (n = 59, p = 0.048), while in squamous cell carcinoma (n = 58), no relationship between PDCD4 expression and clinicopathological parameters was established. These data suggest that the loss of PDCD4 expression is a prognostic factor in lung cancer and may correlate with tumour progression. Copyright

Involvement of programmed cell death 4 in transforming growth factor-β1-induced apoptosis in human hepatocellular carcinoma

The programmed cell death 4 (PDCD4) gene was originally identified as a tumor-related gene in humans and acts as a tumor-suppressor in mouse epidermal carcinoma cells. However, its function and regulatory mechanisms of expression in human cancer remain to be elucidated. We therefore investigated the expression of PDCD4 in human hepatocellular carcinoma (HCC) and the role of PDCD4 in human HCC cells. Downregulation of PDCD4 protein was observed in all HCC tissues tested compared with corresponding noncancerous liver, as revealed by Western blotting or immunohistochemical staining. Human HCC cell line, Huh7, transfected with PDCD4 cDNA showed nuclear fragmentation and DNA laddering characteristic of apoptotic cells associated with mitochondrial changes and caspase activation. Transforming growth factor-β1 (TGF-β1) treatment of Huh7 cells resulted in increased PDCD4 expression and occurrence of apoptosis, also concomitant with mitochondrial events and caspase activation. Transfection of Smad7, a known antagonist to TGF-β1 signaling, protected cells from TGF-β1-mediated apoptosis and suppressed TGF-β1-induced PDCD4 expression. Moreover, antisense PDCD4 transfectants were resistant to apoptosis induced by TGF-β1. In conclusion, these data suggest that PDCD4 is a proapoptotic molecule involved in TGF-β1-induced apoptosis in human HCC cells, and a possible tumor suppressor in hepatocarcinogenesis.

Induction of PDCD4 tumor suppressor gene expression by RAR agonists, antiestrogen and HER-2/neu antagonist in breast cancer cells. Evidence for a role in apoptosis.

The growth of human breast tumor cells is regulated through signaling involving cell surface growth factor receptors and nuclear receptors of the steroid/thyroid/retinoid receptor gene family. Retinoic acid receptors (RARs), members of the steroid/thyroid hormone receptor gene family, are ligand-dependent transcription factors, which have in vitro and in vivo growth inhibitory activity against breast cancer cells. RAR-agonists inhibit the proliferation of many human breast cancer cell lines, particularly those whose growth is stimulated by estradiol (E2) or growth factors. Additionally, RAR-agonists and synthetic retinoids such as Ferentinide have been shown to induce apoptosis in malignant breast cells but not normal breast cells. To better define the genes involved in RAR-mediated growth inhibition of breast cancer cells, we used oligonucleotide microarray analysis to create a database of genes that are potentially regulated by RAR-agonists in breast cancer cells. We found that PDCD4 (programmed cell death 4), a tumor suppressor gene presently being evaluated as a target for chemoprevention, was induced about three-fold by the RARα-selective agonist Am580, in T-47D breast cancer cells. RAR pan-agonists and Am580, but not retinoid X receptors (RXR)-agonists, stimulate the expression of PDCD4 in a wide variety of retinoid-inhibited breast cancer cell lines. RAR-agonists did not induce PDCD4 expression in breast cancer cell lines, which were not growth inhibited by retinoids. We also observed that antiestrogen and the HER-2/neu antagonist, Herceptin (Trastuzumab), also induced PDCD4 expression in T-47D cells, suggesting that PDCD4 may play a central role in growth inhibition in breast cancer cells. Transient overexpression of PDCD4 in T-47D (ER+, RAR+) and MDA-MB-231 (ER−, RAR−) cells resulted in apoptotic death, suggesting a role for PDCD4 in mediating apoptosis in breast cancer cells. PDCD4 protein expression has previously been reported in small ductal epithelium of normal breast. To date, there has been no report of induction of PDCD4 expression by RAR-agonists, antiestrogen or HER2/neu antagonist in breast cancer cells and its potential role in apoptosis in these cells.

β1-integrin protects hepatoma cells from chemotherapy induced apoptosis via a mitogen-activated protein kinase dependent pathway

β1‐integrin modulates cellular phenotype by mediating signals from the extracellular matrix (ECM). Although overexpression of integrin molecules in hepatocellular carcinoma (HCC) has been reported, the role of overexpressed β1‐integrin in the disease process of HCC is not fully understood. The authors investigated the effects of β1‐integrin on apoptosis in hepatoma cells.

Novel human PDCD4 (H731) gene expressed in proliferative cells is expressed in the small duct epithelial cells of the breast as revealed by an anti-H731 antibody.

The novel gene H731 (approved name: PDCD4 (programmed cell death 4)) has been isolated as an antigen gene of the monoclonal antibody Pr‐28 which recognized a nuclear antigen in proliferating cells. The gene is homologous to the mouse gene (MA‐3/Pdcd4/A7–1) which was associated with apoptosis and was shown to suppress tumor promoter‐induced neoplastic transformation. A polyclonal antibody against H731‐protein derived from an extract of Escherichia coli transformed with an H731 expression plasmid was prepared, and the H731‐protein expression in human normal and tumor cells using the antibody was studied. The staining patterns of asynchronous cultures of human normal fibroblasts (MRC‐5) were heterogeneous but the antigen was accumulated in the nuclei at the G0 phase. On the contrary, the antigen was overproduced and localized in the cytoplasm during the cell cycle in tumor cell lines. Immunohistological studies revealed that the H731‐protein was highly expressed in bladder carcinoma and breast carcinoma tissues compared with the normal tissues so far tested. These results indicated that expression of the H731‐protein was up‐regulated or induced in the proliferative cells. Immunohistological studies also revealed that the protein was abundantly expressed in the small duct epithelial cells of the normal mammary gland.

Up-regulation of PDCD4 in senescent human diploid fibroblasts

Programmed cell death 4 (PDCD4) has a common MI domain sharing with death associated protein 5 (DAP5) and a component of eukaryotic translation initiation factor (eIF4G) complex and it might also work as a tumor suppressor. We could find that the message and product of Pdcd4 gene were up-regulated in senescent human diploid fibroblasts. In yeast two hybrid analysis, the C-terminal region of PDCD4 interacted with ribosomal protein S13 (RPS13), ribosomal protein L5 (RPL5), and TI-227H. In in vitro binding assay, RPS13, a component of 40S ribosome was stably bound to PDCD4. We also found that PDCD4 was localized to polysome fractions. We could pull out eIF4G with GST-PDCD4, but eIF4E did not interact with PDCD4. From these results, we could assume that PDCD4 might regulate the eIF4G-dependent translation through direct interactions with eIF4G and RPS13 in senescent fibroblasts.

Primary and secondary culture of rat ameloblasts in serum-free medium

SummaryEnamel is the hardest tissue in vertebrates. Ameloblasts are derived from epithelial cells and are responsible for enamel formation. They secrecte enamel matrix components in which amelogenins are the major proteins, the biochemical properties of which are well known. However, little is known about the characteristics of ameloblasts themselves or about the functions of amelogenins. In this study, we developed a novel primary and secondary culture system for ameloblasts using a monoclonal antibody which recognized amelogenin (En3). The cell layer on dentine removed from rat mandibular incisors was isolated and cultured in low calcium, serum-free medium. Primary culture was performed on collagen-coated culture plates and typically, two types of cells appeared. One major type changed morphology after the addition of a high concentration of calcium to the medium. Expression of amelogenin was shown as cytoplasmic particles in these cells using En3. In the secondary culture, expression of amelogenins was also observed. In this system, the cells grew and maintained the expression of amelogenin for about 3 weeks.

Assignment of the programmed cell death 4 gene (PDCD4) to human chromosome band 10q24 by in situ hybridization.

H731 (PDCD4) was isolated from a human glioma cDNA library by screening with a monoclonal antibody, Pr-28, which recognizes a nuclear component of proliferating cells (Matsuhashi et al., 1997). It has been reported that H731-like (197/15a) cDNA was identical to H731 except for having an additional 84 bp in the 5)-region resulting in a different predicted start codon, and its expression was regulated by cytokines in NK cells (Azzoni et al., 1998). A mouse homologue was associated with apoptosis (MA-3: Shibahara et al., 1995; Pdcd4: Jurisicova et al., 1998), and was shown to suppress tumor promoter-induced neoplastic transformation (A7-1: Cmarik et al., submitted). The expression was suppressed by topoisomerase inhibitors (TIS: Onishi and Kizaki, 1996). Materials and methods

Transforming growth factor‐β1‐induced apoptosis is blocked by β1‐integrin‐mediated mitogen‐activated protein kinase activation in human hepatoma cells

Growth factors and extracellular matrices cooperatively regulate cellular behavior. However, the interactions between transforming growth factor‐β1 (TGF‐β1) and integrins in hepatic cells are not fully understood. We investigated the effects of β1‐integrin on TGF‐β1‐regulated growth of hepatoma cells. Human hepatoma cell lines HepG2, Huh7, and Hep3B were stably transfected with β1‐integrin, and the parental, and mock‐ and β1‐integrin‐transfected hepatoma cells were treated with TGF‐β1. Modulation of apoptosis and pathways involved in the process were investigated. TGF‐β1 suppressed the growth of hepatoma cells, and apoptosis was observed in Hep3B and Huh7. Hepatoma cells transfected with β1‐integrin were protected from TGF‐β1‐induced apoptosis. Mitogen‐activated protein (MAP) kinase inhibitors, PD98059, SB203580, and SP600125, abolished this protective effect of β1‐integrin, but herbimycin A and wortmannin were ineffective. Hepatoma cells overexpressing β1‐integrin showed increased activities of MAP kinases, and TGF‐β1 induced sustained activation of MAP kinases in these cells, but only transient activation in mock‐transfected cells. These data suggest that MAP kinases activated by β1‐integrin provide a strong anti‐apoptotic signal during TGF‐β1‐induced apoptosis in human hepatoma cells. Therefore β1‐integrin‐mediated signals may contribute to the development and progression of hepatocellular carcinoma.

Expression patterns of programmed cell death 4 protein in normal human skin and some representative skin lesions

Abstract:  Expression of a tumor suppressor gene, programmed cell death 4 (PDCD4), was investigated at the protein level in the human skin. Immunohistochemically, PDCD4 protein expressed mainly in suprabasal layers, while PDCD4‐positive and ‐negative areas were observed discontinuously in the basal cell layer of the epidermis. In hair follicles, the suprabulbar area including the hair and inner root sheath was immunoreactive, while the bulbar area, containing germinative cells which were strongly proliferating cell nuclear antigen (PCNA)‐positive, was not or less. PDCD4 therefore appears to be important in the differentiation of hair follicles. PDCD4‐positive cells were localized in the inside layers while PCNA‐positive cells were located in the basal layer in the outer root sheath of hair follicles. The cells of sebaceous glands and sweat glands also were PDCD4‐positive. The PDCD4 protein was localized mostly in nuclei of cutaneous cells. PDCD4 expression was found to be suppressed in the epidermis overlying an adult T‐cell lymphoma (ATL), possibly reflecting a paracrine effect of factors produced by ATL cells. PDCD4 expression was suppressed in the keratinocyte cell line HaCaT by exposure of cultures to epidermal growth factor, transforming growth factor‐β1 or hepatocyte growth factor. Immunohistochemically, various skin cancers tended to show less PDCD4 expression than normal skin. Promotion of expression might prove useful in preventing or treating certain skin cancers.