[Acidophil stem cell pituitary neuroendocrine tumors/adenoma: a clinicopathological analysis of five cases].

Objective: To investigate the clinicopathological characteristics of acidophil stem cell pituitary neuroendocrine tumors (PitNET)/adenoma. Methods: Five cases of acidophil stem cell PitNET/adenoma were diagnosed between May 2022 and July 2023 at the Second Hospital of Hebei Medical University, Shijiazhuang, China. The clinicopathological features of the tumor were analyzed by using histology, immunohistochemistry, and electron microscopy. The relevant literature was reviewed. Results: There were 1 male and 4 females, aged from 23 to 69 years. Patient 3 was 55 years old at the time of diagnosis and first surgery, and relapsed 5 years later. The patients' median age was 32 years. Patients 1 and 5 showed elevated blood prolactin, with various degrees of hormonal symptoms except Patient 3, who showed only tumor compression symptoms. Imaging studies showed that all cases involved the sellar floor. The tumors of Patients 1, 2 and 5 were closely related to the cavernous sinus segment of the internal carotid artery. The tumors exhibited a diffuse growth pattern with chromophobic to slightly acidophilic cytoplasm. A few of tumor cells showed chromophobic cytoplasm. The nucleoli were conspicuous. Intranuclear inclusion bodies and variably-sized clear vacuoles were observed occasionally. Under electron microscope, marked mitochondrial abnormalities were observed, including increased mitochondria number, expanded hypertrophy, and absence of mitochondrial ridge fracture. Some mitochondrial matrices were dense, while some were vacuolated. Conclusions: Acidophil stem cell PitNET/adenoma is a rare type of pituitary adenomas/PitNETs. It often has a more clinically aggressive manner with immature cells, diffuse expression of PIT1, prolactin, and varying degrees of growth hormone expression. Because of the obvious diversity of their clinical hormone status and hormone immune expression, the diagnosis of this type tumor is still a challenge.

[Analysis of risk factors for hemorrhage in patients with acute poisoning treated with hemoperfusion].

Objective: To explore the clinical characterist ics and risk factors of hemorrhage complicated by hemoperfusion therapy in patients with acute poisoning. Methods: In January 2021, the clinical data of 196 patients with acute poisoning who received hemoperfusion therapy in the Second Affiliated Hospital of Air Force Military Medical University from January 2018 to December 2020 were analyzed, and the patients were divided into bleeding group and non-bleeding group according to whether the patients were complicated with bleeding. Multivariate logistic regression was used to analyze the independent risk factors for hemorrhage in patients treated with hemoperfusion. Results: A total of 21 patients in the bleeding group and 175 patients in the non-bleeding group were included. There was no significant difference in general data such as gender, age, and body mass index between the two groups (P>0.05) . Organophosphorus pesticides (χ(2)= 4.56, P=0.030) , HA230 perfusion device (χ(2)=4.12, P=0.042) , platelet count (t=-2.33, P=0.009) and activated partial thromboplastin time (t=14.53, P<0.001) at 2 h of perfusion were the influencing factors of hemorrhage in patients with acute poisoning treated with hemoperfusion. Among them, organophosphorus pesticides, 2 h perfusion activated partial thromboplastin time ≥35 s and other factors were independent risk factors forcomplicated bleeding (P<0.05) . Conclusion: Patients with acute poisoning, especially organophosphorus pesticide poisoning, are at greater risk of bleeding during hemoperfusion therapy. Monitoring of changes in activated partial thromboplastin time should be strengthened and the dose of anticoagulants should be adjusted in time to reduce the risk of bleeding.

[Factors affecting the recovery of cholinesterase activity in patients with acute chlorpyrifos poisoning].

Objective: To investigate the related factors affecting the recovery of cholinesterase (ChE) activity in patients with acute chlorpyrifos poisoning. Methods: In February 2020, the clinical data of acute chlorpyrifos poisoning patients admitted in our hospital from January 2016 to December 2019 were retrospectively analyzed. The outcome variable was the time of ChE activity recovered to 50% lower limit of normal value, and multivariate linear regression analysis was performed to explore its influencing factors. Results: A total of 78 patients, 43 males and 35 females, with an average age (39.58±14.77) years were enrolled in this study. The average time of serum ChE activity recovered to 50% lower limit of normal value was (24.45±2.64) days. There was a correlation between hemoperfusion (r=-0.644) , atropine dosage (r=0.498) , chlorophosphorus dosage (r=0.432) and the time of serum ChE activity recovered to 50% lower limit of normal value, in which hemoperfusion was significantly negatively correlated with the time of serum ChE activity recovered to 50% lower limit of normal value (ß=-4.222, P<0.05) . Conclusion: The recovery of serum ChE activity in patients with acute chlorpyrifos poisoning is very slow. Hemoperfusion can quickly remove chlorpyrifos, its metabolites and inflammatory mediators in the blood, thus effectively promoting the recovery of ChE activity.

Identification and validation of a hub gene prognostic index for hepatocellular carcinoma.

Aims: We aim to provide new insights into the mechanisms of hepatocellular carcinoma (HCC) and identify key genes as biomarkers for the prognosis of HCC. Materials & methods: Differentially expressed genes between HCC tissues and normal tissues were identified via the Gene Expression Omnibus tool. The top ten hub genes screened by the degree of the protein nodes in the protein-protein interaction network also showed significant associations with overall survival in HCC patients. Results: A prognostic model containing a five-gene signature was constructed to predict the prognosis of HCC via multivariate Cox regression analysis. Conclusion: This study identified a novel five-gene signature (CDK1, CCNB1, CCNB2, BUB1 and KIF11) as a significant independent prognostic factor.

Lay abstract Given the poor success of traditional treatments in improving the prognosis of hepatocellular carcinoma (HCC), we need new techniques to improve survival. The new techniques must be checked for accuracy and we must assess whether we can utilize it to achieve individualized treatment. The finding of this study, which examined genetic differences between tumor tissues and normal tissues, is that patients with a high-risk genetic 'signature' have worse results and a shorter survival time than those with a low-risk profile. We first screen hub genes related to the survival status of HCC patients. Then we construct a risk score model to predict the prognosis of HCC and confirm that the model is highly credible. It is reasonable to believe that our risk score model has a high predictive value for the prognosis of HCC.

NLRP6-caspase 4 inflammasome activation in response to cariogenic bacterial lipoteichoic acid in human dental pulp inflammation.

AIM: To explore the presence and function of NLRP6-caspase 4 inflammasome in human pulp tissue and human dental pulp cells (HDPCs). METHODOLOGY: Pulp tissue was collected from freshly extracted human caries-free third molars and third molars with irreversible pulpitis. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were performed to assess the expression of NLRP6-caspase 4 inflammasome. HDPCs were prepared from normal human pulp tissues and challenged with Porphyromonas gingivalis LPS. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were performed to assess if LPS can upregulate NLRP6 and caspase-4. HDPCs were further challenged with LPS followed with cytosolic Streptococcus mutans lipoteichoic acid (LTA). SiRNA targeting NLRP6 and Casp4 and pharmacology inhibitor Ac-FLTD-CMK and MCC950 were used to assess if Streptococcus mutans LTA can activate the NLRP6 but not the NLRP3 inflammasome. Western blot and ELISA were performed to evaluate inflammasome activation. The Student's t-test and one-way anova were used for statistical analysis. RESULTS: NLRP6-caspase 4 inflammasome was upregulated and activated in inflamed human dental pulp tissue. In HDPCs, Porphyromonas gingivalis LPS upregulated the expression of NLRP6, CASP1 and CASP4 in a type I interferon dependent manner. After LPS priming, cytosolic Streptococcus mutans LTA triggered NLRP6-caspase 4 inflammasome activation. Knockdown of NLRP6 or CASP4 using siRNA or using pharmacology inhibitor Ac-FLTD-CMK but not MCC950 efficiently suppressed inflammasome activation by cytosolic LTA. CONCLUSIONS: NLRP6-caspase 4 inflammasome may play an important role in pulp inflammation and immune defence. Inflammatory caspases represent a pharmacological target to restrain pulpal inflammation.

[Effects of early supplement of exogenous L-carnitine on renal function in severely scalded rats].

Objective: To explore the effects of early exogenous L-carnitine supplementation on renal function in severely scalded rats. Methods: According to the random number table, sixty-six adult female Sprague-Dawly rats were divided into healthy control group (n=6), scald alone group (n=30), and scald+ carnitine group (n=30). In the latter two groups, the rats were inflicted with full-thickness scald of 30% total body surface area on the back, and the lactated Ringer's solution was injected through the tail vein for resuscitation immediately after scald. At post injury hour (PIH) 1, rats in scald+ carnitine group were intraperitoneally injected with 100 mg/mL L-carnitine solution 400 mg/kg, while rats in scald alone group were intraperitoneally injected with the same volume of normal saline. Rats in these two groups were injected once every 24 hours thereafter. Six rats were taken from each of scald alone group and scald+ carnitine group to collect the renal tissue and abdominal aorta blood at PIH 6, 12, 24, 48, and 72, respectively. The serum content of total protein, albumin, urea nitrogen, creatinine, and cystatin C were determined by the automatic biochemical analyzer. Renal tissue was stained with hematoxylin-eosin to observe histopathological changes. Rats in healthy control group did not undergo any treatment, and their renal tissue and blood sample were extracted and analyzed in the same way as those of severely scalded rats. Data were statistically analyzed with one-way analysis of variance and Bonferroni method. Results: (1) The serum content of total protein and albumin of rats in scald alone group at each time point after injury was significantly lower than that in healthy control group (P<0.05). The serum content of total protein of rats in scald+ carnitine group was significantly higher than that in scald alone group at PIH 12 and 24 (P<0.05), and the serum content of albumin of rats in scald+ carnitine group was significantly higher than that in scald alone group at PIH 12 (P<0.05). The serum content of total protein and albumin of rats in scald alone group and scald+ carnitine group showed a trend of decrease followed by an increase, with the lowest value at PIH 24. (2) The serum content of urea nitrogen and creatinine of rats in scald alone group at each time point after injury was significantly higher than that of healthy control group (P<0.05). The serum content of urea nitrogen of rats in scald+ carnitine group was significantly lower than that in scald alone group at PIH 6, 48, and 72 (P<0.05). The serum content of creatinine of rats in scald+ carnitine group was significantly lower than that in scald alone group at PIH 12, 24, 48, and 72 (P<0.05). The serum content of urea nitrogen and creatinine of rats in scald alone group and scald+ carnitine group showed a trend of increase followed by a decrease, with the peak value at PIH 12. (3) The serum content of cystatin C of rats in scald alone group at PIH 6, 12, 24, 48, and 72 was (0.250±0.030), (0.330±0.070), (0.300±0.060), (0.240±0.060), and (0.190±0.030) mg/L, and the content at the first 4 time points were significantly higher than (0.170±0.020) mg/L of healthy control group (P<0.05). At PIH 24, the serum content of cystatin C of rats in scald+ carnitine group was (0.210±0.040) mg/L, which was significantly lower than that of scald alone group (P<0.05). The serum content of cystatin C of rats in scald alone group and scald+ carnitine group showed a trend of increase followed by a decrease, with the peak value at PIH 12. (4) The renal tissue of rats in healthy control group was almost normal, and the degree of renal tissue injury of rats in scald+ carnitine group was lighter than that in scald alone group at each time point after injury. At PIH 24, the renal tissue of rats in scald alone group showed extensive swelling of the renal tubular epithelial cells, vacuolar degeneration and necrosis, loss of brush borders, and nuclear shrinkage; more than 2/3 of the renal tubular cell nuclei disappeared, the tubular lumen was narrowed, necrotic exfoliated cells could be seen in the lumen, and edema and inflammatory cell infiltration could be seen in the renal interstitial. Compared with those of scald alone group, significantly reduced severity of edema and necrosis of renal tubular epithelial cells, as well as less inflammatory cell infiltration were observed in the renal tissue of rats in scald+ carnitine group. Conclusions: Early supplement of L-carnitine in severely scalded rats can reduce the damage of renal cells, accelerate the restoration of the content of total protein, albumin, urea nitrogen, creatinine, and cystatin C, thereby maintaining the stability of renal function metabolism level.

[Effect of early supplementation of exogenous carnitine on liver mitochondrial damage in severely scalded rats and its pathological mechanism].

Objective: To observe the effect of early supplementation of exogenous carnitine on liver mitochondrial damage in severely scalded rats and to explore its pathological mechanism. Methods: Seventy-two adult female Sprague-Dawley rats were divided into sham injury group, scald injury group, and scald injury+ carnitine group according to the random number table, with 24 rats in each group. Rats in sham injury group was sham injured on the back by immersing in 37 ℃ water bath for 12 s without fluid replacement. While rats in scald injury and scald injury+ carnitine groups were inflicted with 30% total body surface area (TBSA) full-thickness scald on the back by immersing in 98 ℃water bath for 12 s. Immediately after injury, rats in scald injury group and scald injury+ carnitine group were injected with Ringer's lactate solution with the dosage of 4 mL·kg(-1)·%TBSA(-1) via tail vein according to the Parkland formula, meanwhile rats in scald injury+ carnitine group were injected with L-carnitine solution with dosage of 300 mg·kg(-1)·d(-1) via tail vein from post injury hour (PIH) 1. At PIH 12, 24, 48 and 72, abdominal aorta blood and liver tissue were collected from 6 rats in each group. The serum levels of carnitine, ß-hydroxybutyric acid, and ornithine carbamoyltransferase (OCT) were determined with enzyme-linked immuno sorbent assay, and the serum levels of lactate dehydrogenase (LDH), alanine aminotransferase(ALT), and aspartate transaminase (AST) was determined by automatic biochemical analyzer, Pathological changes of rats liver tissue were detected with HE staining. Data were processed with analysis of variance of factorial design and Student-Newman-Keulstest or Tamhane test, Bonferroni correction. Results: (1) Compared with sham injury group, the serum level of carnitine of rats in scald injury group was significantly lower at each time point (P<0.05), and that of scald injury+ carnitine group was significantly lower at PIH 12, 24, and 48 (P<0.05). The serum level of carnitine of rats in scald injury+ carnitine group at PIH 72 [(28.2±3.0) µg/mL] was similar to that in sham injury group[(29.4±4.0) µg/mL, P>0.05]. The serum level of carnitine in scald injury+ carnitine group was significantly higher than that in scald injury group at each time point (P<0.05). (2) The serum levels of ß-hydroxybutyric acid of rats in scald injury group and scald injury+ carnitine group were significantly lower than those in sham injury group at each time point (P<0.05). The serum levels of ß-hydroxybutyric acid of rats in scald injury and scald injury+ carnitine groups both showed a trend of increase, and they peaked at PIH 72 [(1.77±0.30) , (2.93±0.44) mmol/L, respectively]. The serum levels of ß-hydroxybutyric acid in scald injury+ carnitine group were significantly higher than those of scald injury group at each time point (P<0.05). (3) The serum levels of OCT of rats in scald injury and scald injury+ carnitine groups were significantly higher than those of sham injury group at each time point (P<0.05). The serum levels of OCT of rats in scald injury group and scald injury+ carnitine groups both showed a trend of decrease, and they peaked at PIH 12 [(186.28±6.77), (163.38±9.34) ng/mL, respectively]. The serum levels of OCT of rats in scald injury+ carnitine group were significantly lower than those of scald injury group at each time point (P<0.05). (4) Compared with those of sham injury group, the serum levels of LDH of rats in scald injury group were significantly higher at each time point (P<0.05). Compared with those of sham injury group, those of scald injury+ carnitine group were significantly higher at PIH 12 and 24 (P<0.05), which peaked at PIH 12 [(2 226±274) U/L]. The serum levels of LDH of rats in scald injury+ carnitine group were close to those of sham injury group at PIH 48 and72 (P>0.05). The serum levels of LDH of rats in scald injury+ carnitine group were significantly lower than those of scald injury group at each time point (P<0.05). (5) The serum levels of ALT and AST of rats in scald injury group and scald injury+ carnitine group were significantly higher than those of sham injury group at each time point (P<0.05). In scald injury+ carnitine group, the serum levels of ALT of rats were significantly lower than those in scald injury group at PIH 48 and 72 (P<0.05), and the serum level of AST of rats was significantly lower than that in scald injury group at PIH 48 (P<0.05), and the serum levels of AST and ALT of rats were close to those in scald injury group at other time points (P>0.05). The serum levels of ALT and AST in scald injury+ carnitine group both showed a trend of decrease, and they peaked at PIH 12 [(260±25), (1 511±145) U/L, respectively]. (6) The liver tissue of rats in sham injury group was basically normal at each time point. The degree of liver injury of rats in scald injury+ carnitine group was lighter than that in scald injury group. The liver tissue of rats in scald injury group at PIH 72 showed obvious cytoplasm loose, liver tissue structure loss with diffuse fatty degeneration and large coagulative necrosis. Only partially scattered fatty degeneration was observed in the liver tissue of ras in scald injury+ carnitine group. Conclusions: By early supplementation of exogenous carnitine, serum levels of carnitine and ß-hydroxybutyric acid can be restored to normal levels faster, alleviate mitochondrial damage of hepatocytes, and maintain the metabolic stability of hepatocytes in early stage of severe scald.

[Identification of a new pro-invasion factor in tumor microenvironment: progress in function and mechanism of extracellular ATP].

Up to 90% of all cancer related morbidity and mortality can be attributed to metastasis. In recent years the study of tumor microenvironment, its cellular and molecular components, and how they can affect neoplastic progression toward metastasis, has become a hot focus in cancer research. Accumulated evidence shows that the formation of metastasis is a multi-step sequential process, in which, the tumor cells continuously interact with the host microenvironment. Host derived factors, i.e. growth factors/inhibitors, angiogenic factors, chemokines, etc. together with different types of host cells, play important roles in the tumor progression towards metastasis. The interaction between the tumor cells and host microenvironment determines the fate of metastasis. The reveal of this interaction mechanism provides us an opportunity to find effective mode of interference and develop novel anti-metastasis drugs. In this review, we have summarized our work on a new pro-invasion factor identified in tumor microenvironment and how it affects tumor invasion and metastass. Adenosine triphosphate (ATP), the key intracellular energy currency, accumulates within the tumor microenvironment and is closely involved in cancer cell metabolism and in antitumor immunity. The established role of ATP as a growth modulator and a proinflammatory mediator endues ATP and other purines with potential players in host-tumor interaction. Our study demonstrated that extracellular ATP stimulated human cancer invasion in in vitro tests. Increased migration and invasive ability across Matrigel was observed in some human carcinoma cell lines, including the prostate, breast, colon, melanoma and lung, when stimulated with ATP or its analogues. ATP enhanced the motility of cancer cells via increasing the amount and length of lamellipodia and filopodia, which were necessary for the cell motility. Significant increase in Rac1 and Cdc42 activities was observed. Using cDNA microarray we found that the expression of a panel of invasion/metastasis-related genes was significantly changed, including the increased expression of interleukin (IL)-8 and matrix metalloproteinase-3 (MMP-3) after ATP treatment. Changes of some epithelial-mesenchymal transition (EMT)-related factors were also observed, including the increase of snail, decrease of E-cadherin and claudin-1. Multiple P2Y receptors subtypes were expressed on tumor cells, but P2Y2 and P2X7 receptors were found to be mainly responsible for the pro-invasive effect of ATP. Down-regulation of either P2Y2 or P2X7 abolished ATP effect on cancer invasion and expression of EMT/invasion-related genes. Further, we found that P2Y2 receptor trans-activated with epidermal growth factor receptor (EGFR) and co-activated extracellular regulated protein kinases (ERK1/2) signaling pathway, which was involved in regulating expression of EMT and other related genes. In nude mice experiment, the pro-invasive effect of ATP was further confirmed. In summary, our results reveal that ATP is a potential pro-invasive factor in tumor microenvironment. P2Y2/P2X7 receptors act as a mediator in the regulation of ATP-induced EMT and invasion of cancer cells. Given that tumor microenvironment is rich in ATP and other purines, we hypothesize that ATP might be a potential invasion stimulator in tumor microenvironment. Blocking ATP receptor might be a therapeutic target on cancer.

[Association of single nucleotide polymorphisms in PAI-1 with breast cancer susceptibility and prognosis].

OBJECTIVE: To study the effects of single nucleotide polymorphisms (SNP) in Plasminogen activator inhibitor 1(PAI-1) on breast cancer susceptibility and patients' prognosis among a Chinese Han women population. METHODS: Six tag SNP (tSNP) of PAI-1 were selected according to HapMap CHB population, and TaqMan realtime PCR method was used to genotype the 6 tSNP in 1 160 breast cancer cases and 1 318 age-matched controls among Chinese Han women. Haplotypes and diplotypes were inferred according to genotyping data and linkage disequilibrium. Finally, the associations of tSNP, haplotypes and dipltypes with breast cancer susceptibility and patients' prognosis were analyzed. RESULTS: Regarding to breast cancer susceptibility, for rs6090 (G>A), AA genotype carriers had 3.79 times higher risk of developing breast cancer (OR=4.79, 95%CI=1.01-22.64, P=0.048 0) than GG or GA genotype carriers. For rs2227672 (G>T), TT genotype carriers had 1.52 times higher breast cancer risk than GG or GT genotype carriers (OR=2.52, 95%CI=1.26-5.01, P=0.008 6). Regarding to breast cancer prognosis, women who carried rs2227692 (C>T) CT genotype had 46% lower risk of developing recurrence, metastasis or death than CC genotype carriers (HR=0.54, 95%CI=0.30-0.97, P=0.040 4). Using stratified association analysis, among BMI<23 patients, those women who carried AA genotype of rs2227631 (G>A) had 3.99 times higher risk of developing the events (recurrence, metastasis or death) than GG or GA genotype carriers (HR=4.99, 95%CI=1.66-15.02, P=0.004 2). Among HER2 positive patients, those women who carried AA genotype of rs2227667 (G>A) had 2.98 times higher risk of developing the events (recurrence, metastasis or death) than GG or GA genotype carriers (HR=3.98, 95%CI=1.47-10.80, P=0.006 7). Among patients with tumors>2 cm, those women who carried rs2227692 (C>T) CT or TT genotype had 51% lower risk of developing the events (recurrence, metastasis or death) than CC genotype carriers (HR=0.49, 95%CI=0.27-0.88, P=0.017 0). CONCLUSIONS: The study indicates that single nucleotide polymorphisms in PAI-1 may affect breast cancer susceptibility and survival in Chinese Han women. The study may contribute to individualized evaluation of breast cancer risk and patients' prognosis if these data are validated in some other Chinese Han populations.

P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells.

BACKGROUND: Our previous study demonstrated that extracellular adenosine 5'-triphosphate (ATP) stimulated prostate cancer cell invasion via P2Y receptors. However, the purinergic receptor subtype(s) involved in this process remains unclear. Here we aimed to determine whether P2Y2, one subtype of P2Y receptors, was involved in the invasion and metastasis of prostate cancer cells, and elucidated the underlying mechanism. METHODS: RNAi was introduced to silence the expression of P2Y2. In vitro invasion and migration assays and in vivo experiments were carried out to examine the role of P2Y2 receptor in cell invasion and metastasis. cDNA microarray was performed to identify the differentially expressed genes downstream of ATP treatment. RESULTS: P2Y2 was significantly expressed in the prostate cancer cells. Knockdown of P2Y2 receptor suppressed cell invasion and metastasis in vitro and in vivo. Further experiments identified that ATP could promote IL-8 and Snail expression and inhibit E-cadherin and Claudin-1 expression. Knockdown of P2Y2 receptor affected the expression of these EMT/invasion-related genes in vitro and in vivo. CONCLUSION: P2Y2 receptor promotes cell invasion and metastasis in prostate cancer cells via some EMT/invasion-related genes. Thereby, P2Y2 receptor could be a potential therapeutic target for the treatment of prostate cancer.

Antisense epidermal growth factor receptor RNA transfection in human glioblastoma cells down-regulates telomerase activity and telomere length.

Epidermal growth factor receptor is overexpressed and/or amplified in up to 50% of glioblastomas, suggesting an important role of this gene in glial tumorigenesis and progression. In the present study we demonstrated that epidermal growth factor receptor is involved in regulation of telomerase activity in glioblastoma. Antisense-epidermal growth factor receptor approach was used to inhibit epidermal growth factor receptor expression of glioblastoma U87MG cells. Telomerase activity in antisense-epidermal growth factor receptor cells decreased by up to 54 folds compared with control cells. Moreover, the telomere lengths of antisense-epidermal growth factor receptor cells were shortened. In addition, the tumorigenicity of antisense-epidermal growth factor receptor cells was significantly inhibited. Taken together, there were strong correlations between tumorigenicity and epidermal growth factor receptor expression levels, and between tumorigenicity and telomerase activity. These results provide evidence that epidermal growth factor receptor plays an important role in the regulation of telomerase activity of glioma cells. Our findings provide new insights into both the biological functions of epidermal growth factor receptor and the regulation of telomerase activity. The inhibition of telomerase activity triggered by antisense-epidermal growth factor receptor treatment may reflect yet another mechanism of antisense-epidermal growth factor receptor approach in tumour suppression.

Isolation and identification of poly-alpha-(1-->4)-linked 3-O-methyl-D-mannopyranose from a hot-water extract of Mycobacterium vaccae.

A polysaccharide around 3.6 kDa has been identified as the major carbohydrate moiety of a antineoplastic protein-polysaccharide complex (PS4A) obtained by boiling intact cells of Mycobacterium vaccae in water. 1H and 13C NMR spectra of this polysaccharide suggested it was a highly homogeneous polymer composed substantially of one monomer, probably an alpha-linked O-methylated mannose. Comparison of the COSY spectra of the original and acetylated polymer indicated that the glycosidic linkage and the methyl ether were interchangeable, at O-3 and O-4. Further study demonstrated that the benzyolated hydrolysate of the polymer was 1,2,4,6-tetra-O-benzoyl-3-O-methyl-beta-mannopyranose. The hydrolysate was 3-O-methyl-alpha, beta-mannopyranose and the polymer was therefore poly-alpha-(1-->4)-linked 3-O-methyl-D-mannopyranose. This conclusion was further confirmed with an authentic sample of the monomer, which had spectral data identical to those of the hydrolyzate and co-eluted from an ion-exchange HPLC with the major sugar in the hydrolysate.

Establishment and characterization of a human cell line from paediatric cerebellar glioblastoma multiforme.

Permanent glioma cell lines are invaluable tools in understanding the biology of glioblastomas. The present study reports the establishment of a clonal human cell line, GBM6840, derived from a biopsy of paediatric cerebellar glioblastoma multiforme. GBM6840 had a doubling time of 32 h and grew as a monolayer of large round cells that retained immunopositivity for glial fibrillary acidic protein and vimentin. Karyotypic analysis revealed a modal chromosome number of 68 and polysomies of chromosomes 3, 5 and 20, as well as the presence of 3-4 marker chromosomes. GBM6840 also showed anchorage-independent growth in soft agar and tumour formation in nude mice. The p16(CDKN2A) gene was transcriptionally silenced by hypermethylation, consistent with the lack of protein expression observed in the original tumour and cultured cells. Western blot analysis revealed normal protein expression of pRb and CDK4. It appears that p16 is the major component altered in the cell cycle pathway and may confer these cells unrestrained proliferation potential. Neither EGFR gene amplification nor over-expression of the protein was detected in the cultured cells. Over-expression of the p53 protein was observed in the majority of cells, despite undetectable mutation (exons 5-8) in the gene. One allele of the PTEN gene was found to be mutated during in vitro cultivation. Telomerase activity was demonstrated in the cultured cells but not in the original tumour, supporting the hypothesis that telomerase is required for the in vitro immortalization process.

Altered expression of the suppressors PML and p53 in glioblastoma cells with the antisense-EGF-receptor.

Gene amplification and enhanced expression of the epidermal growth factor receptor (EGFR) represent the major molecular genetic alteration in glioblastomas and it may play an essential role in cell growth and in the carcinogenic process. On the other hand, the nuclear suppressor proteins PML and p53 are also known to play critical roles in cancer development and in suppressing cell growth. Here we report that, in glioblastoma cells with defective EGFR function, the expressions of both promyelocytic leukaemia (PML) and p53 were altered. Cells that were transfected with the antisense-cDNA of EGFR were found to have more cells in G1 and fewer cells in S phase. In addition, the transfected cells were found to be non-responsive to EGF-induced cell growth. Interestingly, the expression of the suppressors p53 and PML were found to be significantly increased by immunohistochemical assay in the antisense-EGFR cells. Moreover, the PML expression in many of the cells was converted from the nuclear dot pattern into fine-granulated staining pattern. In contrast, the expressions of other cell cycle regulated genes and proto-oncogene, including the cyclin-dependent kinase 4 (cdk4), retinoblastoma, p16INK4a and p21H-ras, were not altered. These data indicate that there are specific inductions of PML and p53 proteins which may account for the increase in G1 and growth arrest in antisense-EGFR treated cells. It also indicates that the EGF, p53 and PML transduction pathways were linked and they may constitute an integral part of an altered growth regulatory programme. The interactions and cross-talks of these critical molecules may be very important in regulating cell growth, differentiation and cellular response to treatment in glioblastomas.

Isolation and biological activities of an antineoplastic protein-polysaccharide complex (PS4A) obtained from Mycobacterium vaccae.

A mixture of water-soluble protein-polysaccharides (PS4A) was isolated by boiling intact cells of Mycobacterium vaccae, a fast growing mycobacterium. Sephadex G-75 column chromatography of the crude extract separated the biologically active high molecular weight (> 50 kDa) fraction (in the void volume) from the low molecular weight degradation products. Compositional analysis demonstrated that PS4A contained protein and polysaccharide in a ratio of approximately 1.5 to 1, but no lipids were detected. The antineoplastic activity was tested in vivo by a S-180 murine sarcoma model using female CFW mice. The immunostimulating activity was tested in vitro using murine peritoneal macrophages isolated from BALB/C mice. The results demonstrated that PS4A significantly decreased tumor incidence in vivo and produced activation of murine peritoneal macrophages. However, the antineoplastic activity was only attributable to the high molecular weight fraction of the protein-polysaccharide complex. The low molecular weight fraction had no antineoplastic activity in vivo despite stimulation of TNF-alpha production in vitro. In vitro experiments also demonstrated that although all PS4A components significantly increased TNF-alpha production by macrophages, the high molecular weight fraction stimulated more IL-1 production, indicating a better immunostimulating activity.

Restoration of wild-type PTEN expression leads to apoptosis, induces differentiation, and reduces telomerase activity in human glioma cells.

PTEN is a candidate tumor suppressor gene identified on human chromosome 10q23.3 that is frequently mutated or deleted in 30% to 44% of glioblastomas. Transient expression study of PTEN in glioma cells indicates that PTEN plays an important role in cellular proliferation, tumorigenicity, cell migration, and focal adhesions. In this study, we examined the biological consequences on U87MG glioma cells after stable gene transfer of wild-type PTEN. Cells stably expressing wild-type PTEN protein were found to have suppressed proliferation, as determined by cell counting and Ki-67 staining, as well as inhibited anchorage-independent growth. The PTEN-expressing cells also showed higher expression of glial fibrillary acidic protein and changed morphologically from spindle-shaped to elongated cell bodies with multiple slender processes, suggesting that these cells have undergone differentiation. In addition, telomerase activity decreased more than 10-fold in PTEN-expressing cells when compared with control cells. More importantly, apoptosis was detected in about 5% of PTEN-expressing cells, representing a 17-fold (p < 0.01) increase over the control cells. Taken together, these results suggest that PTEN plays an important role in regulation of cell homeostasis by maintaining a balance between proliferation, differentiation, and apoptosis.

Formulation and biological activity of antineoplastic proteoglycans derived from Mycobacterium vaccae in chitosan nanoparticles.

Although heat-killed suspensions of Mycobacterium vaccae have been tested clinically against tuberculosis and cancer, from a pharmaceutical perspective it would be advantageous to utilize isolated active components rather than the heat-degraded bacterial materials. In our laboratory we have isolated from M. vaccae a number of high-molecular-weight proteoglycans with considerable immunological and antineoplastic activity. The structure of one of these, PS4A, obtained by extraction with boiling water, seems to consist of a basic unit with a 20-kDa protein core to which are attached glucans and O-methylated 4-kDa polysaccharides. The molecular weight is (approx.) 50 kDa, but because of self-association, that of the recovered high-molecular-weight fraction is greater than 150 kDa. A similar, but even larger, molecule (PS4alpha, MW approximately 20 MDa) is obtained by cold extraction with 8 M urea. Both are active in-vivo against an S-180 murine sarcoma model but have no activity in-vitro, suggesting an antitumour effect involving activated macrophages. For this reason gelatin nanoparticles are unsuitable as a vehicle but chitosan seemed to be a promising alternative. In this report we describe the production of stable 600-700-nm diameter nanoparticles of chitosan without organic solvents. Adsorption and release of bovine serum albumin seemed to be affected by the charge of the two reactants and at high doses not all adsorbate was released. PS4A, because of structural and compositional differences, had to be loaded on to the chitosan by freeze drying a suspension of the nanoparticles in a solution of the drug. After a rapid (burst) release phase, the rate of release into water was steady for the next 4 h, but not all the drug was released. In-vivo it was evident that PS4A and PS4alpha were equally active in solution or when formulated in the chitosan nanoparticles. These results show that chitosan nanoparticles, readily prepared without the use of organic solvents, are a suitable vehicle for the delivery of these immunostimulants from M. vaccae; the formulations might find application as antitumour agents.

A study on the relationship between antisense EGFR cDNA fragments and nuclear matrix proteins in glioblastoma cells.

The association of antisense epidermal growth factor receptor (EGFR) cDNA fragments with nuclear matrix from EGFR-antisense transfected glioblastoma cell lines U343 and U87 was investigated. A 1015 bp DNA fragment (primer I-II) was amplified in both genomic DNA and nuclear matrix-associated DNA (NM DNA) from EGFR-antisense transfected glioblastoma cell lines U343E and U87E. Two different DNA fragments (940 bp and 110 bp) were amplified by primer I-III in both genomic DNA and NM DNA of U343E, while one 110 bp PCR product was shown with the same primer in both genomic DNA and NM DNA of U87E only. After EGFR-antisense transfection, the binding property of the 110 bp DNA fragment (primer IV-V) to nuclear matrix was not affected. Southwestern blotting demonstrated the presence of antisense EGFR cDNA binding nuclear matrix proteins. Our findings demonstrate that not only EGFR DNA is associated with nuclear matrix, but the transfected antisense EGFR cDNA also binds to nuclear matrix proteins. The nuclear matrix is most likely involved in the replication and transcription of antisense EGFR cDNA or hybridisation with sense mRNA in vitro.

Antisense epidermal growth factor receptor RNA transfection in human malignant glioma cells leads to inhibition of proliferation and induction of differentiation.

The epidermal growth factor receptor (EGFR) is a protooncogene that is frequently observed with alterations in late stage gliomas, suggesting an important role of this gene in glial tumorigenesis and progression. In this study we evaluated an antisense EGFR approach as an alternative therapeutic modality for glioblastomas. We transfected U-87MG cells with an antisense EGFR construct and obtained several clones stably expressing lower or undetectable levels of EGFR protein. These clones were found to have impaired proliferation as well as a reduced transforming potential to grow in soft agarose. The number of cells positive for the cell cycle-specific nuclear antigen Ki-67 was also significantly decreased (P < 0.05) in antisense EGFR-transfected clones compared with parental or empty vector-transfected cells. Flow cytometric analysis revealed that the proportion of cells in G0/G1 phases of the cell cycle in the antisense clones increased by up to 31% compared with control cells, whereas the proportion of cells in S phase decreased by up to 58%. In addition, the antisense EGFR-transfected cells showed higher expression of glial fibrillary acidic protein and a more differentiated form, with smaller cell bodies possessing fine tapering cell processes. These results suggest that EGFR plays a major role in modulating cell growth and differentiation in glioblastoma cells. Our experimental model of antisense EGFR provides a basis for future development of antisense EGFR oligodeoxynucleotides in treatment of glioblastomas.