Release of fibroblast growth factor-1 by human squamous cell carcinoma correlates with autocrine cell growth

Summary A squamous cell carcinoma cell line Nakata proliferated in serum-free culture and was not responsive to exogenous fibroblast growth factor-1 (FGF-1). Immunostaining revealed that Nakata cells expressed FGF-1 in their cytoplasms and nuclei. Two molecular mass species of FGF-1 (16 and 18 kDa) were identified in cell extracts by Western blot. These cells also expressed high-affinity FGF-1 binding sites (Kd=360 pM, 28 000 sites/cell). The results of cross-linking with [¹²⁵I]FGF-1 demonstrated the presence of two bands with molecular masses of 160 and 140 kDa. The addition of FGF-1 specific antisense oligonucleotides at 25 μM to Nakata cells resulted in an 82% inhibition in cell growth and suppressed FGF-1 expression. This effect was dose-dependent and specific, because sense oligonucleotides were ineffective in inhibiting cell growth. In addition, Nakata cell growth was suppressed by an anti-FGF-1 neutralizing antibody, which resulted in a 52% inhibition at 8 μg/ml. These results demonstrate that Nakata cells produce FGF-1, and indicate that this growth factor acts in an autocrine manner by interacting with FGF-1 binding sites on Nakata cells.     

Variable X chromosome inactivation patterns in near-tetraploid murine EC × somatic cell hybrid cells differentiatedin vitro

For the cytogenetic study of X chromosome inactivation as an X chromosome dosage compensation mechanism, we isolated a number of XXXX, XXX, and XXY near-tetraploid mouse hybrid cell clones by fusing XX or XO embryonal carcinoma cells with lymphocytes carrying a structurally altered X chromosome(s). The inactive X chromosome from the female lymphocyte was reactivated in these hybrid clones which retained embryonal carcinoma morphology so far as they were cultured on the collagen-coated plastic surface in the medium supplemented with leukemia inhibitory factor (LIF) and betamercaptoethanol (BME). Some of these clones developed balloon-like cystic embryoid bodies when they were allowed to form cell aggregates in medium without LIF and BME in bacteriological petri dishes to which they do not adhere. X chromosome inactivation occurring during this process detected by the incorporation of 5-bromodeoxyuridine did not conform to the expected pattern leaving two X chromosomes active in every tetraploid cells. This may suggest either that the X-inactivation mechanism evolved primarily, for the diploid cell is unable to deal with tetraploid conditions efficiently, or that the present system ofin vitro differentiation represents an anomalous situation never encounteredin vivo.     

Correlation of the expression of double-stranded RNA-dependent protein kinase (p68) with differentiation in head and neck squamous cell carcinoma

p68 is an inducible protein kinase which is believed to be an important factor in the regulation of both viral and cellular protein synthesis. We have produced a monoclonal antibody (TJ4C4) which specifically detects p68, and which can be used to detect this antigen in formalin-fixed, paraffin-embedded tissues. Because p68 plays an important role in cellular protein synthesis, we hypothesized that it may correlate with normal and neoplastic cellular differentiation. One hundred and seventy-seven head and neck squamous cell carcinoma specimens, representing 82 patients, were studied. The relative amount, frequency, and distribution of p68 expression were determined by microscopic evaluation of ABC immunoperoxidase-stained specimens. A spectrum of immunoreactivity was detected in 156 of 177 tumors, as well as within the normal squamous epithelium. Normal, actively proliferating cells, such as the basal layer of squamous epithelium, expressed comparatively little p68. Increased p68 expression was noted to parallel the morphologic features of cellular differentiation. In neoplastic tissue, p68 expression also increased with the degree of cellular differentiation. These data demonstrate that the expression of p68 parallels the degree of cellular differentiation in squamous cell carcinoma of the head and neck region, as well as within normal squamous mucosa. Therefore, p68 may provide an objective biologic measure of cellular differentiation which does not depend on morphologic features.     

Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.     

GPD1L inhibits renal cell carcinoma progression by regulating PINK1/Parkin-mediated mitophagy

Few approaches have been conducted in the treatment of renal cell carcinoma (RCC) after nephrectomy, resulting in a high mortality rate in urological tumours. Mitophagy is a mechanism of mitochondrial quality control that enables selective degradation of damaged and unnecessary mitochondria. Previous studies have found that glycerol-3-phosphate dehydrogenase 1-like (GPD1L) is associated with the progression of tumours such as lung cancer, colorectal cancer and oropharyngeal cancer, but the potential mechanism in RCC is still unclear. In this study, microarrays from tumour databases were analysed. The expression of GPD1L was confirmed by RT–qPCR and western blotting. The effect and mechanism of GPD1L were explored using cell counting kit 8, wound healing, invasion, flow cytometry and mitophagy-related experiments. The role of GPD1L was further confirmed in vivo. The results showed that GPD1L expression was downregulated and positively correlated with prognosis in RCC. Functional experiments revealed that GPD1L prevented proliferation, migration and invasion while promoting apoptosis and mitochondrial injury in vitro. The mechanistic results indicated that GPD1L interacted with PINK1, promoting PINK1/Parkin-mediated mitophagy. However, inhibition of PINK1 reversed GPD1L-mediated mitochondrial injury and mitophagy. Moreover, GPD1L prevented tumour growth and promoted mitophagy by activating the PINK1/Parkin pathway in vivo. Our study shows that GPD1L has a positive correlation with the prognosis of RCC. The potential mechanism involves interacting with PINK1 and regulating the PINK1/Parkin pathway. In conclusion, these results reveal that GPD1L can act as a biomarker and target for RCC diagnosis and therapy.     

Determination of Ki67 Growth Fraction and Oestrogen Receptors In Screen-Detected Breast Cancer Using Cytological Preparations

Oestrogen receptor (ER) status of 77 cases of screen-detected breast cancer has been determined using cytological preparations. In 48% ER status was positive, which was the same proportion as that formed in a control group of age-matched patients with symptomatic breast carcinoma. Since the screen-detected group contained more low grade tumours, the percentage of ER-positive cases would be expected to be higher. the reasons for the discrepancy are discussed. Ki67 score has been determined for 41 cases of screen-detected cancer. Ki67 score showed a positive correlation with histological tumour grade and a negative correlation with ER status. However, there was no correlation with tumour size or lymph node status. the Ki67 scores in the screen-detected cancers were essentially similar to those found in an age-matched symptomatic group, but the very low scores were only found in the screened group.     

Augmentation of antitumor immunity in regional lymph nodes by local immunotherapy

We have previously reported that the antitumor effect of OK-432, a streptococcal preparation, is markedly augmented when injected intratumorally together with fibrinogen (OK-432/fbg) [1]. In order to elucidate the effects of this immunotherapy on regional lymph nodes (RLN), we carried out both morphological and functional analyses of the RLN from colonic cancer patients treated with OK-432/ fbg. Computer-aided morphometry revealed that the maximal cross-sectional areas and the broadest diameters of the RLN were significantly greater (p<0.01) in patients who had undergone local immunotherapy than in patients who had not. The component structures of RLN, such as sinus, follicle and paracortex, were all enlarged in the OK-432/fbg-treated patients, and necrosis of metastatic tumors was observed. RLN lymphocytes recovered from OK-432/fbg treated patients showed elevated reactivity to phytohemagglutinin (PHA) and the stimulation index was clearly higher than that of control patients. Flow cytometric analysis revealed a predominance of T-cells, especially CD4 subsets, and higher positivity for both CD25 and HLA-DR. Furthermore, RLN lymphocytes killed more effectively K562 and Daudi cells in the patients who had had immunotherapy. These results suggest that the effect of local immunotherapy with OK-432/fbg is not restricted to the site of injection but extends to the lymph nodes, and contributes to tumor regression through the augmentation of cellular immunity.Abbreviations RLN regional lymph node(s) - OK-432/fbg OK-432/fibrinogen solution - PHA phytohemagglutinin - NK natural killer - LAK lymphocyte activated killer     

核纤层蛋白B1通过影响端粒酶活性调控肝癌细胞生长

核纤层蛋白B1(nuclear lamina protein B1,LMNB1)高表达于肝癌组织中,通过敲低LMNB1探讨其对肝癌细胞增殖的影响及其机制。利用siRNA在肝癌细胞中敲低LMNB1,Western blotting检测敲低效果,使用端粒重复序列扩增法(telomeric repeat amplification protocol assay,TRAP)检测其端粒酶活性变化。利用实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,qPCR)检测其端粒长度变化。并通过CCK-8、克隆形成、Transwell、划痕实验检测其生长,侵袭和迁移能力变化。利用慢病毒系统构建稳定敲低LMNB1的HepG2细胞,检测其端粒长度及端粒酶活性变化,采用SA-β-gal衰老染色检测细胞衰老情况,通过裸鼠皮下成瘤实验及对肿瘤后续的组化染色,SA-β-gal衰老染色,端粒荧光原位杂交(fluorescence in situ hybridization,FISH)检测其对成瘤性的影响。最后利用生物信息分析的方法寻找LMNB1在临床肝癌组织中的表达情况,及其与临床分期、病人生存期的关系。HepG2和Hep3B中敲低LMNB1后端粒酶活性显著降低,细胞增殖、迁移和侵袭能力显著降低,细胞和裸鼠成瘤实验证明稳定敲低LMNB1后端粒酶活性降低的同时端粒长度缩短,细胞发生衰老,此外细胞成瘤性降低,Ki-67表达降低,生物信息分析结果显示,LMNB1高表达于肝癌组织,且与肿瘤分期和患者生存相关。LMNB1在肝癌细胞中过表达,其有望成为评估肝癌患者临床预后的指标和精准治疗的靶点。