Vascular neointimal hyperplasia, a pathological process observed in cardiovascular diseases such as atherosclerosis and pulmonary hypertension, involves the abundant presence of vascular smooth muscle cells (VSMCs). The proliferation, migration, and autophagy of VSMCs are associated with the development of neointimal lesions. Circular RNAs (circRNAs) play critical roles in regulating VSMC proliferation and migration, thereby participating in neointimal hyperplasia. However, the regulatory roles of circRNAs in VSMC autophagy remain unclear.

We aimed to identify circRNAs that are involved in VSMC autophagy-mediated neointimal hyperplasia, as well as elucidate the underlying mechanisms.

Dual-luciferase reporter gene assay was performed to validate two competing endogenous RNA axes, hsa_circ_0001402/miR-183-5p/FKBP prolyl isomerase like (FKBPL) and hsa_circ_0001402/miR-183-5p/beclin 1 (BECN1). Cell proliferation and migration analyses were employed to investigate the effects of hsa_circ_0001402, miR-183-5p, or FKBPL on VSMC proliferation and migration. Cell autophagy analysis was conducted to reveal the role of hsa_circ_0001402 or miR-183-5p on VSMC autophagy. The role of hsa_circ_0001402 or miR-183-5p on neointimal hyperplasia was evaluated using a mouse model of common carotid artery ligation.

Hsa_circ_0001402 acted as a sponge for miR-183-5p, leading to the suppression of miR-183-5p expression. Through direct interaction with the coding sequence (CDS) of FKBPL, miR-183-5p promoted VSMC proliferation and migration by decreasing FKBPL levels. Besides, miR-183-5p reduced BECN1 levels by targeting the 3'-untranslated region (UTR) of BECN1, thus inhibiting VSMC autophagy. By acting as a miR-183-5p sponge, overexpression of hsa_circ_0001402 increased FKBPL levels to inhibit VSMC proliferation and migration, while simultaneously elevating BECN1 levels to activate VSMC autophagy, thereby alleviating neointimal hyperplasia.

Hsa_circ_0001402, acting as a miR-183-5p sponge, increases FKBPL levels to inhibit VSMC proliferation and migration, while enhancing BECN1 levels to activate VSMC autophagy, thus alleviating neointimal hyperplasia. The hsa_circ_0001402/miR-183-5p/FKBPL axis and hsa_circ_0001402/miR-183-5p/BECN1 axis may offer potential therapeutic targets for neointimal hyperplasia.

Bone morphogenetic protein-7 is (BMP-7) is a potent anti-inflammatory growth factor belonging to the Transforming Growth Factor Beta (TGF-β) superfamily. It plays an important role in various biological processes, including embryogenesis, hematopoiesis, neurogenesis and skeletal morphogenesis. BMP-7 stimulates the target cells by binding to specific membrane-bound receptor BMPR 2 and transduces signals through mothers against decapentaplegic (Smads) and mitogen activated protein kinase (MAPK) pathways. To date, rhBMP-7 has been used clinically to induce the differentiation of mesenchymal stem cells bordering the bone fracture site into chondrocytes, osteoclasts, the formation of new bone via calcium deposition and to stimulate the repair of bone fracture. However, its use in cardiovascular diseases, such as atherosclerosis, myocardial infarction, and diabetic cardiomyopathy is currently being explored. More importantly, these cardiovascular diseases are associated with inflammation and infiltrated monocytes where BMP-7 has been demonstrated to be a key player in the differentiation of pro-inflammatory monocytes, or M1 macrophages, into anti-inflammatory M2 macrophages, which reduces developed cardiac dysfunction. Therefore, this review focuses on the molecular mechanisms of BMP-7 treatment in cardiovascular disease and its role as an anti-fibrotic, anti-apoptotic and anti-inflammatory growth factor, which emphasizes its potential therapeutic significance in heart diseases.

Cardiovascular diseases are the main cause of death worldwide and pose an immense economical burden. In most cases, the underlying problem is vascular occlusion by atherosclerotic plaques. Importantly, different cell types of the vascular wall and the immune system play crucial roles in atherosclerosis at different stages of the disease. Furthermore, atherosclerosis and conditions recognized as risk factors are characterized by a reduced availability of the vasoprotective molecule nitric oxide and an increase in reactive oxygen species, so-called oxidative stress. Transcription factors function as intracellular signal integrators and relays and thus, play a central role in cellular responses to changing conditions. Recent Advances: Work on specific transcriptional regulators has uncovered many of their functions and the upstream pathways modulating their activity in response to reactive oxygen and nitrogen species. Here, we have reviewed for a few selected examples how this can contribute not only to protection against atherosclerosis development but also to disease progression and the occurrence of clinical manifestations, such as plaque rupture.

Transcription factors have pleiotropic outputs and often also divergent functions in different cell types and tissues. Thus, in light of potential severe adverse side effects, a global activation or inhibition of particular transcriptions factors does not seem a feasible therapeutic option.

A further in-depth characterization of the cell- and stage-specific actions and regulation of transcription factors in atherosclerosis with respect to protein-protein interactions and target genes could open up new avenues for prevention or therapeutic interventions in this vascular disease. Antioxid. Redox Signal. 26, 679-699.

The bulbocavernosus (BC) is a sexually dimorphic muscle observed only in males. Androgen receptor knockout mouse studies show the loss of BC formation. This suggests that androgen signaling plays a vital role in its development. Androgen has been known to induce muscle hypertrophy through satellite cell activation and myonuclei accretion during muscle regeneration and growth. Whether the same mechanism is present during embryonic development is not yet elucidated. To identify the mechanism of sexual dimorphism during BC development, the timing of morphological differences was first established. It was revealed that the BC was morphologically different between male and female mice at embryonic day (E) 16.5. Differences in the myogenic process were detected at E15.5. The male BC possesses a higher number of proliferating undifferentiated myoblasts. To identify the role of androgen signaling in this process, muscle-specific androgen receptor (AR) mutation was introduced, which resulted in no observable phenotypes. Hence, the expression of AR in the BC was examined and found that the AR did not colocalize with any muscle markers such as Myogenic differentiation 1, Myogenin, and paired box transcription factor 7. It was revealed that the mesenchyme surrounding the BC expressed AR and the BC started to express AR at E15.5. AR mutation on the nonmyocytic cells using spalt-like transcription factor 1 (Sall1) Cre driver mouse was performed, which resulted in defective BC formation. It was revealed that the number of proliferating undifferentiated myoblasts was reduced in the Sall1 Cre:AR(L-/Y) mutant embryos, and the adult mutants were devoid of BC. The transition of myoblasts from proliferation to differentiation is mediated by cyclin-dependent kinase inhibitors. An increased expression of p21 was observed in the BC myoblast of the Sall1 Cre:AR(L-/Y) mutant and wild-type female. Altogether this study suggests that the nonmyocytic AR may paracrinely regulate the proliferation of myoblast possibly through inhibiting p21 expression in myoblasts of the BC.

Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21.

We have previously shown in separate studies that MDM2 knockdown via antisense MDM2 (AS-MDM2) and E2F1 overexpression via adenoviral-mediated E2F1 (Ad-E2F1) sensitized prostate cancer cells to radiation. Because E2F1 and MDM2 affect apoptosis through both common and independent pathways, we hypothesized that coupling these two treatments would result in increased killing of prostate cancer cells. In this study, the effect of Ad-E2F1 and AS-MDM2 in combination with radiation was investigated in three prostate cancer cell lines: LNCaP cells, LNCaP-Res cells [androgen insensitive with functional p53 and androgen receptor (AR)], and PC3 cells (androgen insensitive, p53(null), and AR(null)). A supra-additive radiosensitizing effect was observed in terms of clonogenic inhibition and induction of apoptosis (caspase-3 + caspase-7 activity) in response to Ad-E2F1 plus AS-MDM2 treatments in all three cell lines. In LNCaP and LNCaP-Res, these combination treatments elevated the levels of phospho-Ser(15) p53 with significant induction of p21(waf1/cip1), phospho-gammaH2AX, PUMA, and Bax levels and reduction of AR and bcl-2 expression. Similarly, AR(null) and p53(null) PC-3 cells showed elevated levels of Bax and phospho-gammaH2AX expression. These findings show that the combination of Ad-E2F1 and AS-MDM2 significantly increases cell death in prostate cancer cells exposed to radiation and that this effect occurs in the presence or absence of AR and p53.

Proteasome-dependent degradation of regulatory proteins is a known mechanism of cell cycle control. p21(WAF1/CIP1) (p21), a negative regulator of the cell division cycle, exhibits proteasome-sensitive turnover and ubiquitination. In the present study, we analyzed the regulatory effects of JNK1 on p21 protein accumulation in p53 null K562 cells. We found that JNK1 (wild type, WT) mediated H(2)O(2)-induced p21 protein up-regulation. Over-expression of JNK1 (WT) could elevate endogenous p21 protein level but did not affect p21 mRNA level and also prolong the p21 half-life as well as inhibited the p21 ubiquitination. These findings indicated that JNK1 could regulate cellular p21 level via inhibiting ubiquitination of p21, which provided a new insight for analyzing the regulatory effect of JNK after stress.

Apoptosis has been suggested to have an important role in the pathogenesis of restenosis in addition to cell migration and proliferation. The aim of the present study was to investigate in an experimental in vivo model the occurrence of apoptosis postangioplasty and its relation to bcl-2 and peroxynitrite detection. Eighteen hypercholesterolemic rabbits underwent transluminal angioplasty of the right iliac artery. The rabbits were sacrificed on the 1st, 2nd, 3rd, 7th, 15th, and 28th day postangioplasty (3 animals per time point) and both the angioplasted and non-injured arteries were studied. Apoptosis was assessed by the terminal uridine nick-end labeling method (TUNEL). Bcl-2 and peroxynitrite were detected by immunochemistry using anti-bcl-2 and anti-nitrotyrosine antibodies. In the angioplasted arteries the number of apoptotic cells was <or=1% of the total cell population in both media and neointima at any of the postangioplasty time points examined. Bcl-2 and nitrotyrosines were detected at all time points in the angioplasted arteries (vs. non-injured, P<0.001), showed similar localization and had the same peaks of expression both in the media (7th day: Bcl-2 66% and nitrotyrosines 74%) and neointima (15th day: Bcl-2 67% and nitrotyrosines 61%). In this experimental model we observed low apoptotic rates while bcl-2 and peroxynitrite products were detected. We can hypothesize that the detection of nitrotyrosines is related with reduced levels of nitric oxide resulting in increased expression of the bcl-2, preventing thus cell death due to either apoptosis or necrosis. Further studies are needed in order to elucidate their role in the restenosis process.

The Cilostazol for RESTenosis (CREST) clinical trial was initiated to evaluate the efficacy of cilostazol, an antiplatelet drug, in inhibiting restenosis after stent implantation in a native coronary artery as evaluated by quantitative coronary angiography. Preliminary results suggest that cilostazol reduces restenosis by 36% over standard therapy alone. Restenosis after coronary stenting is primarily attributed to neointimal formation. Cilostazol decreases the activity of phosphodiesterase type 3, leading to the accumulation of cyclic adenosine monophosphate, which initiates a cascade of events including upregulation of anti-oncogenes p53 and p21 and upregulation of hepatocyte growth factor (HGF). The increase in p53 protein blocks cell cycle progression and induces apoptosis in vascular smooth muscle cells (VSMCs), leading to an antiproliferative effect. Upregulation of local HGF stimulates rapid regeneration of endothelial cells, which inhibits neointimal formation via two mechanisms: inhibition of abnormal VSMC growth and improvement of endothelial function. These mechanisms may be responsible for the improvement in restenosis shown in the CREST trial and a number of other trials in patients who underwent percutaneous transluminal coronary angioplasty. These effects, in addition to antithrombotic and vasodilatory attributes of cilostazol, make it a potentially viable treatment option for preventing restenosis following coronary stenting.

Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCF-mediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.

It has been suggested that genes which exercise checkpoint control during cell cycle traverse are equally important to the process of apoptotic cell death. In this study, we show that the key cell cycle regulatory gene p21(WAF1) is also involved in the execution of apoptosis. p21(WAF1) expression was down-regulated during NaBu-induced apoptosis of senescent normal diploid human 2BS fibroblasts. Conversely, when p21(WAF1) expression was actively suppressed in 2BS cells by a stably transfected antisense p21(WAF1) construct, apoptosis was accelerated and senescence was delayed, as shown by several markers of cell aging. Down-regulation of p21(WAF1) by antisense caused an increase in the phosphorylation and inactivation of pRb. Phosphorylation of pRb was further enhanced upon induction of apoptosis by NaBu. Our results suggest that p21(WAF1), acting through the phosphorylation of pRb, regulates whether 2BS cells cease to proliferate and become senescent but resistant to apoptosis, or whether they accelerate proliferation while becoming more susceptible to apoptotic stimuli.

To examine whether depsipeptide (FK228), a histone deacetylase (HDA) inhibitor, has inhibitory effects on the proliferation of synovial fibroblasts from rheumatoid arthritis (RA) patients, and to examine the effects of systemic administration of FK228 in an animal model of arthritis.

Autoantibody-mediated arthritis (AMA) was induced in 19 male DBA/1 mice (6-7 weeks old); 10 of them were treated by intravenous administration of FK228 (2.5 mg/kg), and 9 were used as controls. The effects of FK228 were examined by radiographic, histologic, and immunohistochemical analyses and arthritis scores. RA synovial fibroblasts (RASFs) were obtained at the time of joint replacement surgery. In vitro effects of FK228 on cell proliferation were assessed by MTT assay. Cell morphology was examined by light and transmission electron microscopy. The effects on the expression of the cell cycle regulators p16INK4a and p21(WAF1/Cip1) were examined by real-time polymerase chain reaction and Western blot analysis. The acetylation status of the promoter regions of p16INK4a and p21(WAF1/Cip1) were determined by chromatin immunoprecipitation assay.

A single intravenous injection of FK228 (2.5 mg/ml) successfully inhibited joint swelling, synovial inflammation, and subsequent bone and cartilage destruction in mice with AMA. FK228 treatment induced histone hyperacetylation in the synovial cells and decreased the levels of tumor necrosis factor alpha and interleukin-1beta in the synovial tissues of mice with AMA. FK228 inhibited the in vitro proliferation of RASFs in a dose-dependent manner. Treatment of cells with FK228 induced the expression of p16INK4a and up-regulated the expression of p21(WAF1/Cip1). These effects of FK228 on p16INK4a and p21(WAF1/Cip1) were related to the acetylation of the promoter region of the genes.

Our findings strongly suggest that systemic administration of HDA inhibitors may represent a novel therapeutic target in RA by means of cell cycle arrest in RASFs via induction of p16INK4a expression and increase in p21(WAF1/Cip1) expression.

Both p16(INK4) and p21(Waf1) are very important negative regulators of the cell cycle. In this study we examined the effects of p21(Waf1) on the transcription of p16(INK4). We determined that p21(Waf1) can activate the transcription of p16(INK4), and that this effect is GC-box dependent. We also found that the transcription factor Sp1 plays a key role in this event. Upregulation of Sp1 contributes to the transcriptional activation and protein level of p16(INK4) mediated by p21(Waf1), and is a potential point of cooperation between the p16/pRb and p14 (ARF)/p53 tumor suppressor pathways.

The identification of novel therapeutic agents for the management of malignant gliomas represents an area of active research. Here, we show that Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; TPL), a stable nitroxide free radical, inhibits the growth of C6 glioma cells both in vitro and in vivo. Morphological features of apoptosis were apparent in C6 cells following in vitro treatment with TPL. Cell death was preceded by dose-dependent increase in p21(WAF1/CIP1) expression, without apparent stabilisation of the TP53 gene product. When C6 cells were grown as xenografts in nude mice, treatment with TPL induced a significant dose-dependent decrease in tumour growth, without signs of general or organ toxicity. Tumours from treated mice showed an increase in the number of apoptotic cells and a decrease in the rate of neo-vascularisation compared with tumours from control mice. Our findings suggest a potential use for TPL as a novel antiproliferative agent for the treatment of malignant gliomas.

Abnormal vascular smooth muscle (VSM) cell proliferation contributes to the development of atherosclerosis and its associated disorders, including angioplasty restenosis. The tumor-suppressor protein p53 has been linked to the development of atherosclerotic lesions, and its homolog, p73, is proving to have contrasting functions in a variety of tissues. As an outgrowth of our previous finding that p73 is increased in serum-stimulated VSM cells and human atherosclerotic tissue, we examined p73 overexpression in VSM cells to elucidate causality of p73 expression with growth response. Overexpression of p73 results in decreased cell cycle transit and is accompanied by apoptosis. The apoptotic changes in p73 overexpressing VSM cells are independent of p53 and are associated with a decrease in levels of p21(waf1/cip1). In conjunction with our previous data finding that p73 is increased in serum-stimulated VSM cells, this work suggests a role for p73 in vascular proliferative diseases.

Normal embryonic development, tissue differentiation and repair in the eukaryote requires a tightly regulated apoptosis, or programmed cell death. Apoptosis also plays an essential role in different pathological processes including atherosclerosis, in which it affects all cell types in the atherosclerotic lesion, including endothelial cells, vascular smooth muscle cells, and macrophages. During atherosclerosis progression, pro- and anti-apoptotic signals abound in the evolving lesion. Apoptosis limits the number of a particular cell type that accumulates in the lesion and slows down the overall progression of the lesion. On the other hand, it contributes to the production of unstable plaques. Many pharmacological agents used to treat cardiovascular and lipid disorders have pro- or/and anti-apoptotic effects. Pharmaceuticals that modulate apoptosis in specific types of cell can potentially serve as anti-atherogenic agents. However, to develop agents for clinical use requires a thorough knowledge of the pathophysiology of apoptosis in atheromatous lesions, a highly cell-specific process. Here we review our current understanding of the process to provide a background for future pharmacological research in the area.

Rheumatoid synovial fibroblasts (RSF) express cyclin-dependent kinase (CDK) inhibitors p16(INK4a) and p21(Cip1) when they are growth-inhibited in vitro. The induction of p16(INK4a) is characteristic of RSF and intra-articular p16(INK4a) gene therapy has been shown to suppress adjuvant arthritis (AA) of rats. The other inducible CDK inhibitor, p21(Cip1), has multiple functions depending on the cell type. They include inhibition of CDK as well as promotion of active CDK complex formation and induction of apoptosis. This study is to discern the biological effects of p21(Cip1) gene transfer into RSF and its therapeutic effects on AA. A recombinant adenovirus containing a human p21(Cip1) gene and control adenoviruses were prepared. RSF infected with these viruses were examined for their cell growth. Apoptotic cell death was evaluated by nuclear staining and DNA fragmentation analysis. In vivo gene therapy of rat AA was carried out by intra-articular injection of the viruses. Severity of the arthritis was clinically scored. The treated joints were examined histologically and proliferating cell nuclear antigens (PCNA) were detected immunohistochemically. The adenoviral p21(Cip1) gene transfer inhibited growth of RSF without inducing apoptosis. p21(Cip1) gene therapy suppressed AA clinically and histologically. The effects were comparable to p16(INK4a) gene therapy. PCNA expression was reduced in the p21(Cip1)-treated joints. The adenoviral gene transfer of p21(Cip1) ameliorated rat AA. The effect was attributable to inhibition of proliferation. Because p21(Cip1) is induced more easily by many chemicals than p16(INK4a), it also appears to be a feasible target in developing anti-rheumatic drugs.

Massive small bowel resection (SBR) increases rates of both enterocyte proliferation and apoptosis. Previous studies have demonstrated increased intestinal expression of proapoptotic bax mRNA and protein, as well as the appearance of an 18-kd bax cleavage product within 12 hours of SBR. This study tested the hypothesis that bax is required for postresection increases in enterocyte apoptosis.

Male bax-null and C57Bl/6 (control) mice underwent either a 50% proximal SBR or sham operation. After 3 days, the remnant ileum was harvested and weighed. Apoptotic indexes, proliferation indexes, villus heights, and crypt depths were determined.

The usual adaptive increases in ileal wet weight, crypt depth, and rate of proliferation occurred in both the control and bax-null mice. Resection significantly increased the rate of apoptosis in the control mice; however, it failed to alter the apoptotic index in the bax-null mice.

Bax is necessary for the increase in apoptosis that occurs after SBR, but its absence has no significant effect on short-term adaptation. These findings suggest that enterocyte proliferation and apoptosis are differentially regulated during intestinal adaptation.

The cyclin-dependent kinase inhibitors interact with cyclin-cdk complexes to arrest mitogen-stimulated transit through the cell cycle, but these proteins have recently been shown to have positive regulatory effects on cyclin-cdk complex activity as well. Most of the previous work in this area has focussed on the finding that overexpressed p21(Waf1/Cip1) causes growth arrest. However, mice lacking p21(Waf1/Cip1) showed normal development with no aberrancy in their cell cycles, and antisense p21(Waf1/Cip1) has only been shown to prevent cell cycle arrest, leading to the conclusion that the cyclin kinase inhibitors may not be required for cell cycle progression. We found that transfection of several lines of vascular smooth muscle cells with antisense oligodeoxynucleotide specific to p21(Waf1/Cip1) correlates with decreased cyclin D1/cdk 4, but not cyclin E/cdk 2, association, yet, unexpectedly, results in dose-dependent inhibition of platelet-derived growth factor-BB-stimulated DNA synthesis and cell proliferation. Our finding that p21(Waf1/Cip1) exhibits permissive effects on growth factor-induced vascular smooth muscle cell cycle progression, such that its presence is required for growth factor-induced proliferation, is the first such report and opens up a fertile area of research relevant to diseases involving vascular cell proliferation.

In pancreatic cancer, the mutation of c-K-ras is a critical event of tumor growth and metastasis. We have previously demonstrated a dominant negative effect of N116Y on the growth of pancreatic cancer cells. To evaluate the potential of N116Y for suppressing the metastatic growth of pancreatic tumor cells, we made a replication-deficient recombinant N116Y adenovirus driven by the carcinoembryonic antigen (CEA) promoter (Ad CEA-N116Y). We demonstrated that the expression of N116Y, growth inhibition, and apoptotic death induction were all specific to pancreatic cancer cell lines (PCI-35 and PCI-43) that were promoter positive, whereas no growth retardation was observed in human embryonic pancreas-derived cell line 1C3D3 after Ad CEA-N116Y infection. We examined the effect of Ad CEA-N116Y on the metastatic growth of PCI-43 colonies in liver, which was generated by tumor injection into the spleen of nude mice. The results showed that Ad CEA-N116Y effectively reduced the number of metastatic colonies without any complication by injecting intrasplenically 5 days after tumor cell inoculation. Thus, N116Y can selectively suppress the metastatic growth of pancreatic tumor cell by using the CEA promoter-driven adenovirus vector indicating that N116Y gene therapy may be potentially useful for the treatment of pancreatic cancer patients with liver micrometastasis.

p21, a potent cyclin-dependent kinase inhibitor, has been known to induce cell cycle arrest in response to DNA-damaging agents. Although p21 has been reported to play an important role in the regulation of apoptosis, the postulated role for p21 in apoptosis is still controversial. Previously, we reported that p21 was induced in a p53-independent manner during ceramide-induced apoptosis in human hepatocarcinoma cell lines. In the present study, we investigated the precise role of p21 in ceramide-induced apoptosis in human hepatocarcinoma cells by using a tetracycline-inducible expression system. Overexpression of p21 by itself did not induce apoptosis in p53-deficient Hep3B cells. However, Hep3B/p21 cells were more sensitive to ceramide-induced apoptosis. In these cells, p21 overexpression did not result in G1 arrest. The expression level of Bax was increased in Hep3B/p21 cells treated with ceramide and its expression was more accelerated under the p21-overexpressed condition compared to that of the p21-repressed condition. Overexpression of Bax induced apoptosis in Hep3B cells. On the other hand, the levels of p21 and Bax protein were increased by ceramide in another hepatocarcinoma cell line, SK-Hep-1, while the Bcl-2 protein level was not changed. Overexpression of Bcl-2 not only suppressed apoptosis but also completely prevented induction of p21 and Bax caused by ceramide in SK-Hep-1 cells. Furthermore, overexpression of p21 antagonized the death-protective function of Bcl-2 and upregulated expression of Bax protein. These results suggest that p21 promotes ceramide-induced apoptosis by enhancing the expression of Bax, thereby modulating the molecular ratio of Bcl-2:Bax in human hepatocarcinoma cells.

p21((WAF1/SDII/CIP1)) (p21) arrests cell growth by inhibiting cyclin-depend kinases. To explore the potential of using p21 for the gene therapy of cervical cancer, we infected human papillomavirus (HPV)-positive cervical cancer cells (HeLa, SiHa, and Z172) and HPV-negative cervical cancer cells (C33A) with recombinant adenovirus encoding p21 cDNA. The results revealed that effective inhibition of cell growth could be achieved by sense p21 adenovirus but not antisense p21 adenovirus infection and occurred through apoptosis as measured by DNA fragmentation and chromatin condensation. Apoptosis was also observed in xenografts of human cervical cancer cells infected with sense p21 adenovirus, as confirmed by in situ terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL). The apoptosis was not prevented by overexpression of the bcl-2 transgene. To sum up, the apoptotic effect suggests that p21 should be a tumoricidal agent instead of a tumoristatic agent in preventing cervical cancers. In addition, our report substantiates the combination of the high efficiency of adenovirus vector-mediated gene delivery and the apoptotic effect of p21.

Adequate control of survival or programmed cell death (apoptosis) of cardiovascular cells appears as an important drug target. While prevention of apoptotic death of cardiomyocytes has been assessed in detail, selective induction of apoptosis of vascular smooth muscle cells or fibroblasts could also be of relevance. Thus, induction of apoptosis of vascular smooth muscle cells by p65 NF-kappa B and Bcl-xL antisense oligonucleotides or p53 overexpression could be useful for limiting vascular lesions associated with restenosis. Although fibroblasts represent the majority of cardiac cells, few attempts were made to induce fibroblast apoptosis in disorders associated with excessive collagen deposition and fibrosis. It is hypothesized that early interference with fibroblast proliferation after myocardial infarction or inflammatory heart disease limits fibrosis which further impairs cardiac performance. A candidate approach could involve growth factor analogues which are known to induce fibroblast apoptosis when an incomplete growth stimulus persists.