Shou Ching Tang

Human BAG-1/RAP46 protein is generated as four isoforms by alternative translation initiation and overexpressed in cancer cells

Previously, a Bcl-2-interacting protein, BAG-1, was cloned from mouse cells and was shown to interact with several other proteins and to be important for inhibition of apoptosis. Human BAG-1 (hBAG-1) cDNA, recently isolated by us and two other groups, has been shown to be identical to a hormone receptor-binding protein, RAP46. However, different molecular masses of hBAG-1 protein products were noted by these three groups. Here we demonstrated that hBAG-1 protein was expressed as four isoforms, designated p50, p46, p33 and p29, with apparent molecular masses of 50 kDa, 46 kDa, 33 kDa and 29 kDa, respectively. Deletion, site-directed mutagenesis and in vitro transcription/translation analysis showed that the four protein products of hBAG-1 were expressed by alternative initiation from four different start codons through a leaky scanning mechanism. Furthermore, we demonstrated that the distinct forms of hBAG-1 have different subcellular localizations, suggesting that they may have distinct functions in the cells. Characterization of hBAG-1 RNA and protein also showed that hBAG-1 was overexpressed in human cervical, breast and lung cancer cell lines. Taken together, these data clarify the conflicting observations reported in the literature and suggest that hBAG-1 is expressed as four forms of protein products, which may play a differential role in apoptosis and oncogenesis of human cells.

The alkaloid sanguinarine is effective against multidrug resistance in human cervical cells via bimodal cell death

Sanguinarine, a benzophenanthrine alkaloid, is potentially antineoplastic through induction of cell death pathways. The development of multidrug resistance (MDR) is a major obstacle to the success of chemotherapeutic agents. The aim of this study was to investigate whether sanguinarine is effective against uterine cervical MDR and, if so, by which mechanism. The effects of treatment with sanguinarine on human papillomavirus (HPV) type 16-immortalized endocervical cells and their MDR counterpart cells were compared. Trypan blue exclusion assays and clonogenic survival assays demonstrated that MDR human cervical cells are as sensitive as their drug-sensitive parental cells to death induced by sanguinarine. Upon treatment of both types of cells with sanguinarine, two distinct concentration-dependent modes of cell death were observed. Treatment with 2.12 or 4.24 microM sanguinarine induced death in most cells that was characterized as apoptosis using the criteria of cell surface blebbing, as determined by light and scanning electron microscopy, and proteolytic activation of caspase-3 and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), as detected by Western blot analysis. However, 8.48 and 16.96 microM sanguinarine caused a second mode of cell death, oncosis, distinguished by cell surface blistering, and neither caspase-3 activation nor PARP cleavage. This study provides the first evidence that sanguinarine is effective against MDR in cervical cells via bimodal cell death, which displays alternative mechanisms involving different morphologies and caspase-3 activation status.

Role of Bcl-2 family proteins and caspase-3 in sanguinarine-induced bimodal cell death

Sanguinarine, a benzophenanthridine alkaloid, has anticancer potential through induction of cell death. We previously demonstrated that sanguinarine treatment at a low level induced apoptosis or programmed cell death (PCD) in the Bcl-2 low-expressing K562 human erythroleukemia cells, and that a high level induced blister cell death (BCD); whereas Bcl-2 overexpressing, sanguinarine-treated JM1 pre-B lymphoblastic cells displayed neither apoptosis nor BCD morphologies. Here, we report that sanguinarine-treated K562 cells, when analyzed by western blot, showed significant increase in expression of the pro-apoptotic Bax protein in apoptosis, but not in BCD. cDNA expression array of PCD in K562 cells failed to reveal the presence of Bax at the gene transcript level, which suggests that this cell death process does not require de novo protein synthesis. Treated JM1 cells, on the other hand, showed an increase in the expression of Bcl-2 protein in both forms of cell death, but failed to show Bax expression. The role of other members of the Bcl-2 family remained negligible. Caspase-3 activation was observed in apoptosis of K562 cells but not in BCD or in sanguinarine-treated JM1 cells. These results suggest that sanguinarine in K562 cells induces apoptosis through increasing Bax and activating caspase-3, whereas sanguinarine-induced BCD involves neither. These results also suggest that in JM1 cells, Bcl-2 may play a role in susceptibility of cells to induction of apoptosis and BCD.

Cloning and characterization of the human BAG-1 gene promoter: upregulation by tumor-derived p53 mutants

BAG-1 is an anti-apoptotic protein that interacts with Bcl-2, Bcl-XL, Hsp70/Hsc70, Raf-1 and numerous hormone or growth factor receptors. Recently, BAG-1 has been found to be overexpressed in a variety of human cancer cell lines and some tumors. However, the molecular mechanism of BAG-1 upregulation is still unclear. In this study, we cloned 0.9 kb of human genomic DNA, BGEV, 5′ flanking the BAG-1 open reading frame. BGEV subcloned into a promoterless luciferase reporter vector conferred high promoter activity in various human cancer cell lines. Deletion analysis of this sequence localized the region of maximal BAG-1 promoter activity from nucleotide positions −353 to −54, upstream of the first start codon CTG. Sequence analysis of the BAG-1 promoter region showed the absence of a TATA box but identified a CCAAT box, several GC boxes, a CpG island and several transcriptional factor binding sites, which may be important in the regulation of BAG-1 transcription. Most importantly, functional characterization of the BAG-1 promoter in vivo demonstrated that gain-of-function p53 mutants derived from human tumors upregulated the transcription of BAG-1 RNA and the expression of a reporter gene from the BAG-1 promoter. These results indicated that we have isolated the functional constitutive BAG-1 promoter. Furthermore, the data suggested that overexpression of BAG-1 in some tumors may be due to upregulation of the human BAG-1 promoter by mutant p53.

Prognostic significance of BAG-1 expression in nonsmall cell lung cancer.

The purpose of this study was to evaluate the expression of BAG‐1 in a cohort of patients with nonsmall cell lung cancer (NSCLC). The intensity and subcellular distribution of BAG‐1 expression were correlated with overall survival. Tumor samples were collected from 85 patients diagnosed with NSCLC between 1993–1995 in St. Johns, Newfoundland. Expression of BAG‐1 was determined by immunohistochemistry using polyclonal anti‐BAG‐1 antibody. There was significant variation in the immunohistochemical staining patterns of BAG‐1, including nonstaining and staining of either the cytoplasm, nucleus or both. Univariate Cox regression analysis showed that those patients whose tumor overexpressed BAG‐1 had a significant reduction in the risk of death (hazard ratio = 0.53, p = 0.03). The survival advantage of patients with BAG‐1 overexpression tumor was also demonstrated by Kaplan‐Meier analysis and log‐rank tests (median survival 30.10 months versus 17.04 months, p = 0.05). In addition, multivariate Cox regression analysis showed that patients whose tumor exhibited intense cytoplasmic staining had a further reduction of the risk of death (hazard ratio = 0.42, p = 0.03) and this effect was independent of age, stage and histology. All stages were included in the analysis. Our preliminary data strongly indicate that further investigation is warranted to better define the role of BAG‐1 as an independent prognostic factor in NSCLC.

Distinct BAG-1 isoforms have different anti-apoptotic functions in BAG-1-transfected C33A human cervical carcinoma cell line.

BAG-1 protein can be expressed as four isoforms of 50, 46, 33 and 29 kDa with different subcellular localizations, which may have different functions in anti-apoptosis, but the exact mechanism remains unclear. We constructed BAG-1 full length and deletion mutated plasmids in a pCR3.1 vector and established stable transfections of BAG-1 isoforms in low BAG-1 expressing C33A cells. Treatment of the transfected cells with cisplatin, staurosporine, paclitaxel and doxorubicine showed that BAG-1 p50, p46 and p33 isoforms enhanced the resistance to apoptosis. BAG-1 p50, p46 and p33 exhibited different degrees of apoptosis inhibition in the transfected cells and BAG-1 p46 isoform had the most pronounced effect on anti-apoptosis. BAG-1 p29 failed to protect the transfected cells from apoptosis. Resistance to apoptosis by BAG-1 isoforms was correlated with decreased caspase-3 activation. We also detected the expression of Bax, Bak, p53, Bcl-2, Bcl-XL, AIF and MRP1 by Western blots. Bcl-2 protein expression was significantly increased in p50, p46 and p33 transfected cells, while the expression of Bax, Bak, p53, Bcl-XL and MRP1 was essentially unchanged. These in vitro results suggest that distinct isoforms of BAG-1 have different anti-apoptotic functions and their functions may be correlated to increased Bcl-2 expression.

Enhanced expression of anti-apoptotic proteins in human papillomavirus-immortalized and cigarette smoke condensate-transformed human endocervical cells: correlation with resistance to apoptosis induced by DNA damage.

Apoptosis plays an important role in various biological processes including embryogenesis, differentiation, homeostasis, and oncogenesis. We have developed a system composed of primary human endocervical cells (HEN), HEN immortalized by human papillomavirus (HPV) type 16, and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). To understand the role of apoptosis in the multistep oncogenesis of human cervical cells, we examined the expression of apoptosis‐associated proteins in our in vitro model system. The results showed no significant difference in the levels of apoptosis‐inducing proteins bak and bax among all the cell types examined. On the other hand, the levels of apoptosis‐inhibiting proteins bcl‐2, bcl‐xL and BAG‐1 increased progressively after immortalization and transformation. The p53 protein level decreased in the HPV16‐immortalized HEN and increased in one of two lines of the CSC‐transformed HEN. Further, the increased levels of apoptosis‐inhibiting proteins in the HPV16‐immortalized and the CSC‐transformed HEN correlated with progressively increased resistance of these cells to apoptosis induced by staurosporine or cisplatin. This study provided the first evidence that overexpression of apoptosis‐inhibiting proteins is important for both multistep oncogenesis and resistance of human endocervical cells to apoptosis induced by DNA‐damaging reagents. Mol. Carcinog. 22:95–101, 1998.

Expression of cellular genes in HPV16-immortalized and cigarette smoke condensate-transformed human endocervical cells.

We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H‐ras, c‐myc, B‐myb, p53, p16INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC‐transformed counterparts of HPV16‐immortalized cells had up‐regulated levels of B‐myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis. J. Cell. Biochem. 66: 309–321, 1997.

Aurintricarboxylic Acid Inhibits Protein Synthesis Independent, Sanguinarine-Induced Apoptosis and Oncosis

Sanguinarine, a benzophenanthridine alkaloid, has anticancer potential through induction of cell death. We previously demonstrated that sanguinarine treatment at a low concentration (1.5 μg/ml) induced apoptosis in K562 human erythroleukemia cells, and a high concentration (12.5 μg/ml) induced the morphology of blister formation or oncosis-blister cell death (BCD). Treatment of cells at an intermediate sanguinarine concentration (6.25 μg/ml) induced diffuse swelling or oncosis-diffuse cell swelling (DCS). To assess the underlying mechanism of sanguinarine-induced apoptosis and oncosis-BCD in K562 cells, we studied their response to pre-treatment with two chemical compounds: aurintricarboxylic acid (ATA) and cycloheximide (CHX). The pretreatment effects of both chemical compounds on apoptosis and oncosis-BCD were evaluated by measuring multiple parameters using quantitative morphology, electron microscopy, terminal deoxynucleotidyl transferase (TdT) end-labeling and annexin-V-binding. ATA, a DNA endonuclease inhibitor, efficiently prevented DNA nicking and inhibited apoptosis almost completely and oncosis-BCD by about 40%, while CHX, a protein synthesis inhibitor, failed to inhibit both apoptosis and oncosis-BCD. These results demonstrate, first, the importance of endonuclease in sanguinarine-induced apoptosis and to some extent in oncosis-BCD and, second, that this inhibition does not require de novo protein synthesis.

Human papillomavirus type 16–immortalized endocervical cells selected for resistance to cisplatin are malignantly transformed and have a multidrug resistance phenotype

Cis‐diamminedichloroplatinum (II) (cisplatin, CDDP) is a highly effective chemotherapeutic agent against cervical cancer, but drug resistance is a major obstacle in its clinical application. The mechanism of drug resistance in human cervical cancer is not well understood. Here, we established an in vitro endocervical, cisplatin‐resistant cell system that mimics the development of cisplatin resistance in the human cervix. Human papillomavirus (HPV) type 16–immortalized human endocervical cells (HEN‐16‐2) were treated with cisplatin, and the cisplatin‐selected cells (HEN‐16‐2/CDDP) were resistant to cisplatin, paclitaxel, actinomycin D, doxorubicin, etoposide, and 5‐fluorouracil, thus demonstrating a multidrug resistance (MDR) phenotype. Furthermore, compared with a similar passage of drug‐sensitive HEN‐16‐2 cells, HEN‐16‐2/CDDP cells exhibited the general growth characteristics of cancer cell lines: faster growth in medium containing serum and high calcium levels, higher saturation density, anchorage‐independent growth, and formation of tumors in nude mice. These results provided the first in vitro evidence that cisplatin selection can transform HPV‐immortalized endocervical cells and cause a phenotype of MDR. Int. J. Cancer 87:818–823, 2000.