Resistance to Fas (APO-1/CD95)-mediated apoptosis and expression of Fas ligand in esophageal cancer: the Fas counterattack

The mechanisms by which esophageal tumors escape immunologic recognition and clearance are only partly understood at the molecular level. Esophageal cancers have been shown to evade host recognition by down-regulation of antigen presentation and production of immunosuppressive factors. Recently, two independent reports have shown that esophageal tumor cells abundantly express Fas ligand (FasL) in vivo. As the triggering agonist for Fas receptor (Fas or APO-1/CD95)-mediated apoptosis of lymphocytes, FasL normally plays immune down-regulatory roles, including activation-induced cell death of T and B cells, as well as maintaining immune privilege in certain organs. Fas ligand expressed by esophageal cell lines has been shown to induce apoptosis of cocultured Fas-sensitive lymphoid cells in vitro. FasL expression by esophageal carcinomas in vivo has been associated with significantly reduced tumor-infiltrating lymphocytes (TILs) in FasL-positive tumor nests, concomitant with significantly increased TIL apoptosis in these nests. These studies support a 'Fas counterattack' mechanism of immune escape in esophageal cancer. By expressing functional Fas ligand, esophageal cancer cells can deplete antitumor lymphocytes by inducing apoptosis. To express functional FasL, esophageal carcinomas also acquire molecular mechanisms to resist autocrine Fas-mediated apoptosis of tumor cells.     

Two-field radical lymphadenectomy in the treatment of esophageal carcinoma

This paper retrospectively compares post-operative complications, mortality and long-term survival of patients with esophageal carcinoma who were treated with standard esophagectomy or with extended two-field lymph node clearance. Fifty-seven patients with resectable esophageal carcinoma were included in the study. Twenty-eight patients were submitted to a radical two-field esophagectomy and lymphadenectomy, while the remaining 29 were submitted to a standard, more conservative, esophagectomy performed mostly through a transhiatal route. The two groups of patients were similar in all clinical, laboratory and pathologic features. There was a significant lower anastomotic leakage rate in the group of patients submitted to a radical lymph node resection; post-operative respiratory complication rate and mortality were similar in both groups. The overall 5-year survival was 20%. When lymph node resection was performed, the 5-year survival rate rose to 36%; it was 44% when nodal involvement was negative and 19% for N1 patients; when standard esophagectomy was the procedure, these figures were 9% (p < 0.05), and 6% respectively.     

Fas ligand and Fas receptor are coexpressed in normal human esophageal epithelium: a potential mechanism of apoptotic epithelial turnover

Fas (CD95/Apo-1) receptor (FasR) is a cell-surface receptor that mediates apoptotic cell death upon triggering by Fas ligand (FasL). We sought to determine whether normal human esophageal epithelial cells express FasL and/or FasR and whether their localization is consistent with a role in the turnover of normal esophageal epithelium. Normal esophageal epithelium was immunohistochemically positive for FasL in upper prickle cell layers and in mature squamous cells, but the proliferative basal layer was negative. FasL mRNA was detected in the same epithelial cell layers by in situ hybridization. Co-localization of FasL mRNA and protein therefore confirmed that FasL expression is induced in esophageal epithelial cells as they reach terminal differentiation. FasR was immunohistochemically detected throughout the esophageal epithelium. Positive TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling) staining confirmed cell death of the FasL and FasR coexpressing mature epithelial cells. CD45-positive immunocytes were notably absent from FasL-expressing upper epithelial layers. The findings are consistent with a contributory role for Fas-mediated autocrine suicide or paracrine fratricide in the apoptotic turnover of normal esophageal epithelium.     

Tumor seeding of the jejunostomy site after transhiatal esophagectomy for esophageal carcinoma

A 65-year-old male patient with squamous cell carcinoma of the esophagus had a transhiatal esophagectomy after a prophylactic tube jejunostomy. The tube was removed 3 weeks after surgery. Ten months later, a painless 2-cm abdominal mass was noted at the previous jejunostomy site. Subsequent segmental resection of the jejunum disclosed metastatic squamous cell carcinoma of the esophagus. It is possible that tumor seeding may develop at the jejunostomy site after transhiatal esophagectomy for esophageal carcinoma.     

Neoadjuvant chemotherapy before surgery for resectable carcinoma of the lower esophagus

Neoadjuvant chemotherapy before surgery has been proposed to improve the outcome in patients with early lower esophageal cancer. To evaluate its effectiveness, we performed a systematic retrospective analysis of consecutive patients treated at the Ottawa Regional Cancer Center with prospective inclusion criteria. Between 1988 and 1992 patients were treated with surgery alone. From 1992 until 1997, patients were uniformly treated with neoadjuvant chemotherapy consisting of cisplatin and 5-fluorouracil. Surgical resection was then performed. Nineteen patients received neoadjuvant chemotherapy and 15 received surgery alone. Although the two arms of the study were balanced for age and sex, there were more patients in the neoadjuvant arm with squamous histology, weight loss and regional nodes at diagnosis. In the neoadjuvant arm, two patients did not have surgery because of progression or toxicity. However, complete resection rates were similar. There was no difference in overall survival or disease-free survival between the two arms (p > 0.4). Multivariate analysis revealed that only the nodal status at diagnosis was predictive of outcome. Neoadjuvant chemotherapy with this regimen does not result in improved survival over surgery alone.     

Attenuation of matrix protein secretion by antisense oligodeoxynucleotides to the cyclin kinase inhibitor p21(Waf1/Cip1)

Progressive fibrosis in major organs, including the heart, the kidney and the vascular tree, plays an important role in mediating chronic disease and atherosclerosis. Production of extracellular matrix proteins, in many cases regulated by the growth factor TGF-beta is an essential component of this process. In a parallel manner to TGF-beta, the cyclin kinase inhibitors (CKIs; which are induced by TGF-beta) regulate transit through the cell cycle, and their effect on growth has been shown to be bimodal in the case of vascular smooth muscle (VSM) cells. Using an antisense oligodeoxynucleotide to the CKI p21(Waf1/Cip1), developed in our laboratory and shown to specifically inhibit p21(Waf1/Cip1) protein levels, we asked whether attenuation of the CKI p21(Waf1/Cip1) by transfection of this oligodeoxynucleotide results in the abolition of TGF-beta-mediated growth inhibition and/or diminished matrix protein production and secretion in the presence or absence of TGF-beta. Specific inhibition of p21(Waf1/Cip1) protein with the antisense oligodeoxynucleotide markedly reduces the production and secretion of the matrix proteins fibronectin and laminin, both in the presence and absence of TGF-beta stimulation, in VSM cells as observed by Western blotting of cell lysate and conditioned medium. In addition, TGF-beta-mediated cell growth inhibition, though attenuated by this oligo, is preserved. Due to the relative ease and safety of transfecting antisense oligodeoxynucleotides into VSM, we believe that this work unmasks a potentially powerful technique for inhibition of matrix protein synthesis in VSM and related cell lines, and may lead to new treatment strategies for atherosclerotic as well as other systemic diseases characterized by aberrant matrix protein secretion.     

CyclinD1、CyclinE、CDK6在原发性肝细胞癌中的表达

探讨细胞周期调控因子CyclinD1、CyclinE、CDK6在原发性肝细胞癌中的表达及意义。应用免疫组织化学、原位分子杂交和细胞图象分析技术检测原发性肝细胞癌组织及其对应的癌旁肝组织(各20例)、正常肝组织(5例)中CyclinD1、CyclinE和CDK6mRNA表达情况。结果显示:CyclinD1、CyclinE和CDK6mRNA在肝细胞癌组织中呈阳性表达,阳性率分别为70%、75%和25%,正常肝组织呈阴性表达;肝细胞癌组织中CyclinD1和CyclinE的阳性表达与癌旁肝组织、正常肝组织相比,差异均有显著性意义(P<0.05);CyclinD1和CyclinE的过表达在肝细胞癌的发生和发展中起着重要作用。     

Changes in p53 and Waf1p21 expression and cell proliferation in esophageal carcinogenesis

AIM: To study the correlation between changes in p53 and Waf1p21 expression and cell proliferation, determined by proliferating cell nuclear antigen (PCNA), at different stages of human esophageal carcinogenesis.METHODS: Biopsied and resected esophageal tissues from a high risk population of esophageal cancer in northern China were used in this study. All specimens were fixed in 85% alcohol and processed for routine histology. The avidin biotin peroxidase complex (ABC) method was used to detect p53, Waf1p21 and PCNA.RESULTS: Strong nuclear staining of p53, Waf1p21 and PCNA was observed in normal esophageal epithelium and epithelia with different lesion severities. As the lesions progressed to dysplasia (DYS) and to esophageal squamous cell carcinoma (SCC), the Waf1p21 immunoreactivity percentage decreased. The number of Waf1p21-positive cells slightly increased from normal to basal cell hyperplasia (BCH), but did not further increase in DYS and SCC. The total number of Waf1p21-positive cells was lower than the number of p53-positive cells in normal and BCH esophageal epithelia and much lower in DYS and SCC. Waf1p21-positive cells were located in the third and fourth cell layers in half of the samples examined, which was 2-4 cell layers higher than the cells expressing PCNA and p53 in the same histological categories of normal, BCH and DYS.CONCLUSION: Low Waf1p21 levels at the DYS stage may be related to a functional loss of p53. Other mechanisms may also be responsible for the decreased Waf1p21 expression in DYS and SCC.     

雄激素受体在脑膜瘤中的表达及其临床意义

目的观察33例脑膜瘤患者肿瘤组织中雄激素受体（AR）的表达,探讨AR的表达与脑膜瘤临床病理的相关性。方法应用免疫组化SP法及原位杂交法检测脑膜瘤组织标本中AR的表达情况。结果AR在恶性脑膜瘤组中的染色强度明显高于良性脑膜瘤组,其平均积分光密度值（IOD）分别是25.41±13.42和6.20±4.75（P〈0.05）;AR mRNA的IOD分别为19.46±9.33和4.72±4.68（P〈0.05）。结论AR在脑膜瘤的表达与肿瘤细胞的病理性质（良、恶性）有关,AR可作为鉴别良恶性脑膜瘤的辅助诊断指标。     

Visualizing the dynamics of p21(Waf1/Cip1) cyclin-dependent kinase inhibitor expression in living animals

Although the role of p21(Waf1/Cip1) gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21(Waf1/Cip1) gene expression in vivo, we attempted to visualize the dynamics of p21(Waf1/Cip1) gene expression in living animals. In this study, we established a transgenic mice line (p21-p-luc) expressing the firefly luciferase under the control of the p21(Waf1/Cip1) gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21-p-luc mice enabled us to monitor the endogenous p21(Waf1/Cip1) gene expression in vivo. By monitoring and quantifying the p21(Waf1/Cip1) gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21(Waf1/Cip1) gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21(Waf1/Cip1) gene expression rise dramatically in benign skin papillomas, suggesting that p21(Waf1/Cip1) plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21(Waf1/Cip1) expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21(Waf1/Cip1) knockout mice, has uncovered a novel role for the p21(Waf1/Cip1) gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes.     

Oncogenic ras induces premature senescence in endothelial cells: role of p21(Cip1/Waf1)

Recent studies have shown that the presence of tumor suppressors such as p53 or p16 account for the lack of transformation in primary cells. To investigate a potential role of active Ras in atherosclerosis, we infected bovine aortic endothelial cells with a replication-deficient, recombinant adenovirus containing the activated H-Ras^61L gene. Ras overexpression led after 72 hours to G1- and G2/M-cell cycle arrest due to induction of p21^Cip1/Waf1. Treatment of Ras-infected endothelial cells with 40 ng/ml TNF-α for 20 hours augmented apoptosis 8-fold in comparison to Ad-Con (control virus with empty expression cassette) infected cells (36.2 % vs. 4.3 %, p < 0.001), while Ras itself did not cause any cell death. Furthermore, more than 58 % of Ras-infected cells stained positive for senescence-associated β-galactosidase activity as opposed to 2 % in control vector-infected cells (p < 0.001), strongly suggesting a senescent phenotype in the Ras-infected population. We found further features of senescence in Ras-transduced endothelial cells, such as growth arrest and the lack of AP-1 serum inducibility. Finally, we evaluated the role of p21^Cip1/Waf1 in this process of senescence. Adenoviral overexpression of p21 led to growth arrest by induction of G1- and G2/M-cell cycle arrest. In addition, p21-overexpressing endothelial cells were highly sensitive for TNF-α induced-apoptosis. Surprisingly, senescence-associated β-galactosidase activity was not apparant in p21-infected endothelial cells, suggesting further signaling events necessary for the senescent morphology of endothelial cells. Our results demonstrate a novel way to render primary endothelial cells senescent by overexpressing oncogenic Ras. Increased sensitivity of senescent endothelial cells for cytotoxic stimuli seemed to be due to Ras-induced upregulation of p21^Cip1/Waf1. Future studies have to investigate a potential role of Ras in human vascular biology. Received: 5 June 2001, Returned for revision: 28 June 2001, Revision received: 6 July 2001, Accepted: 31 July 2001     

Estrogen stimulates expression of p21Waf1/Cip1 in mouse uterine luminal epithelium

p21^Waf1/Cip1 was originally identified as an inhibitor of the cell cycle. Recent evidence suggests that it can act as a positive regulator of the cell cycle under the influence of some growth stimulators. We investigated the effects of ovarian steroids on the expression of p21, DNA synthesis, and mitosis in the uterus. Capsules containing 17β-estradiol (E₂) were subcutaneously implanted in ovariectomized mice that were sacrificed on different days. Their uteri were collected for p21 immunohistochemical staining. To study mitosis and DNA synthesis, colchicine and bromodeoxyuridine (BrdU) were injected into mice 3 or 5 h before sacrifice. The results showed that p21 expression, BrdU incorporation, and the mitotic index in uterine luminal epithelium increased 1 to 2 d after E₂ stimulation and then declined to basal levels between d 3 and 6. Furthermore, cotreatment with progesterone (P₄) and E₂ suppressed both p21 expression and the DNA synthesis stimulated by E₂ alone in uterine epithelial cells. Our results show that estrogen stimulates p21 expression and cell proliferation in uterine luminal epithelium and that cotreatment with P₄ prevents both effects, suggesting that p21 may act as a positive cell-cycle regulator.     

Changes of p53 and Waf1p21 and cell proliferation in esophageal carcinogenesis

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Role of p21(Cip1/Waf1) in cell-cycle exit of endomitotic megakaryocytes.

The cyclin-dependent kinase inhibitor p21(Waf-1/Cip-1) is expressed at high level during megakaryocyte differentiation, but its precise function remains unknown. In this study, it is confirmed that p21 was expressed at a high level in hypoploid (2N and 4N) and polyploid (at least 8N) human megakaryocytes derived from CD34(+) cells. A high expression of p27(Kip1), p16, cyclin E, and cyclin D3 was also found in both populations associated with a hypophosphorylated form of retinoblastoma protein, suggesting that the majority of hypoploid and polyploid megakaryocytes are G(1)-arrested cells. As human megakaryocytes grown in vitro present a defect in their polyploidization, the study switched to the murine model. The modal ploidy of megakaryocytes derived from lineage-negative cells was 32N, and an elevated expression of p21 was found in high-ploidy megakaryocytes. In addition, p21 and p27 were coexpressed in the majority of mature polyploid megakaryocytes. The p21 was detected by immunofluorescence in megakaryocytes derived from p53(-/-) mice, demonstrating a p53-independent regulation during megakaryocyte differentiation. Megakaryocytopoiesis of p21(-/-) mice was subsequently studied. No marked abnormality in the ploidy of primary or cultured megakaryocytes was detected. Overexpression of p21 in p21(-/-) or normal murine megakaryocytes and in human megakaryocytes showed in all these cases a marked inhibition in megakaryocyte polyploidization. In conclusion, while a reciprocal relation is observed between p21 levels in megakaryocytes and the cycling state of the cells, p21 is not essential for the determination of the ploidy profile in normal megakaryocytes in vivo. However, high levels of its expression in cultured megakaryocytes arrest the endomitotic cell cycle.     

The expression of p53, p16, cyclin D1 in esophageal squamous cell carcinoma and esophageal dysplasia]

BACKGROUNDS/AIMS: p53 is known to play a central role in sensing and signaling for the growth arrest and apoptosis in cells with DNA damage. Mutation of p53 is a frequent event in esophageal squamous cell carcinoma (ESCC). p16 protein binds to cyclin dependent kinase 4 (CDK4) inhibiting the ability of CDK4 to interact with cyclin D1, and stimulates the passage through the G1 phase of cell cycle. We observed the expression patterns and frequencies of p53, p16, and cyclin D1 in esophageal dysplasia and in esophageal squamous cell carcinomas. METHODS: In 15 patients of ESCC, 5 patients of esophageal dysplasia and 5 volunteers with normal esophagus, tissue specimens were taken from esophageal lesions during the operation or endoscopic examination. We used specific monoclonal antibodies for p53 protein, p16(INK4 ) protein and cyclin D1. Immunoreactivity was scored. RESULTS: Mean age of all groups was 66 years old (range 47-93) and men to women ratio was 19:1. p53 mutation was observed in 87% (13/15) of ESCC, in 80% (4/5) of esophageal dysplasia, in 0% (0/5) of normal mucosa (p=0.001). p16 expression was seen in 40% (2/5) of esophageal dysplasia, 27% (4/15) of ESCC and 100% (5/5) of normal mucosa (p=0.016). Cyclin D1 expression was not significantly different among 20% (1/5) of esophageal dysplasia, 53% (8/15) of ESCC and 20% (1/5) of normal mucosa. Either the expression of p53 mutation or the loss of p16 occurred in 80% (4/5) of esophageal dysplasia and in 93% (14/15) of ESCC. CONCLUSIONS: The expression of p53 mutation and the loss of p16 might play a central role in the pathogenesis of esophageal squamous cell carcinoma (ESCC), and contribute to the development of precancerous lesion such as dysplasia. In addition, there is a possibility that the mutations of p53 and p16 silencing would be the early events in ESCC development.     

Epidermal growth factor receptor and cyclin D1 are independently amplified and overexpressed in esophageal squamous cell carcinoma

Purpose To assess the status of EGFR, HER-2, and CCND1 at the gene and protein levels in esophageal squamous cell carcinoma.Methods Dual-color FISH assays were performed using DNA probes for EGFR/CEP 7, HER-2/CEP 17, and CCND1/CEP 11. The respective proteins, furthermore, was assessed in IHC assays and correlated with patient and tumor characteristics.Results From 55 ESCCs, 8 (15%) tumors showed gene amplification and 20 (36%) had gene overrepresentation (balanced gene and chromosome 7 polysomy) for EGFR. High-level protein expression was frequent (49%), positively correlated with gene copy numbers (kappa=0.4), and associated with well-differentiated histology (p=0.02). For HER-2, gene amplification was detected in a single tumor (2%) and protein overexpression was rare (9%). CCND1 gene was amplified in 23 (42%) tumors; likewise, CCND1 protein overexpression was common (58%) and prevailed in gene overrepresentation or amplification. Only 1 patient showed gene amplification for both EGFR and CCND1. Survival was not associated with EGFR or CCND1 gene/protein status, whereas negative patients for HER-2 protein had a better survival than positive patients (p=0.04).Conclusions Frequent overexpression and gene amplification of EGFR and CCND1 make these molecules and their pathways potential therapeutic targets for ESCC. In addition, EGFR and CCND1 appeared to be independently altered suggesting alternative mechanisms for pathway activation. Therapeutic agents targeting these molecules are urged to be tested in clinical trials and comprehensive biological analyses should be included to properly interpret the outcome.