Cell cycle regulation of the psoriasis associated gene CCHCR1 by transcription factor E2F1.

The coiled-coil alpha-helical rod protein 1 (CCHCR1) was first identified as a candidate gene in psoriasis and has lately been found to be associated with a wide range of clinical conditions including COVID-19. CCHCR1 is located within P-bodies and centrosomes, but its exact role in these two subcellular structures and its transcriptional control remain largely unknown. Here, we showed that CCHCR1 shares a bidirectional promoter with its neighboring gene, TCF19. This bidirectional promoter is activated by the G1/S-regulatory transcription factor E2F1, and both genes are co-induced during the G1/S transition of the cell cycle. A luciferase reporter assay suggests that the short intergenic sequence, only 287 bp in length, is sufficient for the G1/S induction of both genes, but the expression of CCHCR1 is further enhanced by the presence of exon 1 from both TCF19 and CCHCR1. This research uncovers the transcriptional regulation of the CCHCR1 gene, offering new perspectives on its function. These findings contribute to the broader understanding of diseases associated with CCHCR1 and may serve as a foundational benchmark for future research in these vital medical fields.

Artificial intelligence data-driven 3D model for AIS.

Scoliosis is a 3D deformation of the spinal column, characterized by a lateral deviation of the spine, accompanied by axial rotation of the vertebrae. Adolescent Idiopathic Scoliosis (AIS), is the most common type, affecting children between ages 8 to 18 when bone growth is at its maximum rate. The selection of the most appropriate treatment options is based on the surgeon's experience. So, developing a clinically validated patient-specific model of the spine would aid surgeons in understanding AIS in early stages and propose an efficient method of treatment for the individual patient. This project steps include: Developing a clinically validated patient-specific Reduced Order Finite Element Model (ROFEM) of the spine, predicting AIS progression using data mining and proposing a method of treatment. First we implement FE synergistically with bio-mechanical information, image processing and data science techniques to improve predictive ability. Initial geometry of the spine will be extracted from the x-ray images from different planes and imported to FEM software to generate the spine model and perform analysis. A RO model is developed based on the detailed spinal FEM. Next, a neural network is used to predict the spinal curvature. The ability to predict the severity of AIS will have an immense impact on the treatment of AIS-affected children. Access to a predictive and patient-specific model will enable the physicians to have a better understanding of spinal curvature progression. Consequently, the physicians will be able to educate families, choose the most appropriate treatment option and asses for surgical intervention.

MRI-based radiogenomics analysis for predicting genetic alterations in oncogenic signalling pathways in invasive breast carcinoma.

AIM: To investigate the effect of radiomics in the assessment of alterations in canonical cancer pathways in breast cancer. MATERIALS AND METHODS: Eighty-eight biopsy-proven breast cancer cases were included in the present study. Radiomics features were extracted from T1-weighted sagittal dynamic contrast-enhanced magnetic resonance imaging (MRI) images. Radiomics signatures were developed to predict genetic alterations in the cell cycle, Myc, PI3K, RTK/RAS, and p53 signalling pathways by using hypothesis testing combined with least absolute shrinkage and selection operator (LASSO) regression analysis. The predictive powers of the models were examined by the area under the curve (AUC) of the receiver operating characteristic curve. RESULTS: A total of 5,234 radiomics features were obtained from MRI images based on the tumour region of interest. Hypothesis tests screened 250, 229, 156, 785, and 319 radiomics features that were differentially displayed between cell cycle, Myc, PI3K, RTK/RAS, and p53 alterations and no alteration status. According to the LASSO algorithm, 11, 12, 12, 15, and 13 features were identified for the construction of the radiomics signatures to predict cell cycle, Myc, PI3K, RTK/RAS, and p53 alterations, with AUC values of 0.933, 0.926, 0.956, 0.940, and 0.886, respectively. The cell cycle radiomics score correlated closely with the RTK/RAS and p53 radiomics scores. These signatures were also dysregulated in patients with different oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 statuses. CONCLUSION: MRI-based radiogenomics analysis exhibits excellent performance in predicting genetic pathways alterations, thus providing a novel approach for non-invasively obtaining genetic-level molecular characteristics of tumours.

[Diagnosis and treatment of pancreatic neuroendocrine neoplasmas in Von Hippel-Lindau syndrome].

Von Hippel-Lindau(VHL) syndrome is a rare autosomal dominant hereditary disease, and pancreas is one of the frequently involved intra-abdominal organs, including simple pancreatic cysts, pancreatic serous cystadenomas and neuroendocrine neoplasmas. Most of the VHL-related pancreatic neuroendocrine neoplasmas (VHL-pNEN)were non-functional, but they still have a tendency to be malignant. Treatment options for VHL-pNEN include regular follow-up, surgical resection, and medication therapy. When compared with sporadic pNEN, the malignant degree of VHL-pNEN is lower, with a better prognosis, so the surgical treatment should be carefully considered. The indications of surgery for VHL-pNEN include big primary lesions (≥3 cm), fast tumor doubling time (<500 days), VHL gene mutation on exon 3, malignant manifestations on imaging findings, and functional pNEN lesions. The function-preserving approach should be performed to keep the functional pancreatic parenchyma as much as possible. Even for patients with a late stage malignancy that cannot be radically resected, active medication therapy may still lead to a long-term survival.

CCHCR1 interacts with EDC4, suggesting its localization in P-bodies.

Coiled-coil alpha-helical rod protein 1 (CCHCR1) is suggested as a candidate biomarker for psoriasis for more than a decade but its function remains poorly understood because of the inconsistent findings in the literature. CCHCR1 protein is suggested to be localized in the cytoplasm, nucleus, mitochondria, or centrosome and to regulate various cellular functions, including steroidogenesis, proliferation, differentiation, and cytoskeleton organization. In this study, we attempted to find a consensus between these findings by identifying the interaction partners of CCHCR1 using co-immunoprecipiation with a stable cell line expressing EGFP-tagged CCHCR1. Out of more than 100 co-immunoprecipitants identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the enhancer of mRNA-decapping protein 4 (EDC4), which is a processing body (P-body) component, was particularly found to be the major interacting partner of CCHCR1. Confocal imaging confirmed the localization of CCHCR1 in P-bodies and its N-terminus is required for this subcellular localization, suggesting that CCHCR1 is a novel P-body component. As P-bodies are the site for mRNA metabolism, our findings provide a molecular basis for the function of CCHCR1, any disruption of which may affect the transcriptome of the cell, and causing abnormal cell functions.

Sodium 5,6-benzylidene-L-ascorbate induces oxidative stress, autophagy, and growth arrest in human colon cancer HT-29 cells.

Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT-29 cells with SAA and SBA, and found a significant exposure time-dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC(50) = 5 mM) than SBA (IC(50) = 10 mM). A short-term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase-dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21(+/+) and p21(-/-)), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co-incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N-acetyl-L-cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3-II were found in SBA-treated HT-29 cells; implicating that SBA induced AKT phosphorylation-autophagy and p21-growth arrest in colon cancer HT-29 cells through an NAC-inhibitable oxidative stress pathway.

Legionnaires' disease cluster linked to a metal product aqueous pre-treatment process, Staffordshire, England, May 2008.

In May 2008, a report of two workers from the same construction equipment manufacturing plant who were admitted to hospital with Legionnaires disease confirmed by urine antigen prompted an outbreak investigation. Both cases were middle aged men, smokers, and with no travel, leisure or other common community exposure to Legionella sources. There were no wet cooling towers at the plant or in the surrounding area. No increase in respiratory disease or worker absenteeism occurred at the plant during the preceding month. Wider case ascertainment including alerts to hospitals and medical practitioners yielded no further cases. The environmental investigation (and sampling of water systems for Legionella) identified a Legionella pneumophila serogroup1 (Mab 2b) count of >3.0x10(4)cfu/l in water samples from an aqueous metal pre-treatment tunnel, which generates profuse water aerosol. Drainage, cleaning and biocide treatment using thiazalone eliminated Legionella from the system.

Tumor-specific cytotoxicity and type of cell death induced by sodium 5,6-benzylidene-L-ascorbate.

The cytotoxic activity of sodium 5,6-benzylidene-L-ascorbate (SBA) against eight human cancer cell lines and three human normal cells was investigated, SBA showed slightly higher cytotoxicity against human tumor cell lines, as compared with normal cells, with a tumor-specificity index of 2.0. The human myelogenous leukemia cell lines (HL-60, ML-1, KG-1) were the most sensitive to SBA, followed by human oral squamous cell carcinoma (HSC-2, HSC-3, HSC-4) and human glioblastoma (T98G, U87MG). Human oral normal cells (gingival fibroblast, pulp cell, periodontal ligament fibroblast) were the most resistant. In contrast to actinomycin D, SBA induced little or no activation of caspase-3, caspase-8 and caspase-9 in the HSC-2, HSC-4, T98G and HL-60 cells, regardless of incubation time (either 6 or 24 h). SBA induced little or no internucleosomal DNA fragmentation after 6 h in all of these cells. However, prolonged treatment with SBA (24 h) induced a smear pattern of DNA fragmentation in the HSC-2, HSC-4 and T98G cells and a low level of internucleosomal DNA fragmentation in the HL-60 cells. Electron microscopy demonstrated the destruction of mitochondrial structure and autophagocytosis of broken organelles by SBA in the HSC-2, HSC-4 and HL-60 cells. At higher concentrations of SBA, necrotic cell death was observed in the HSC-2 cells, but not in the T98G cells, where the production of acidic organelles (detected by acridine orange staining) was much lower than that attained by nutritional starvation, a well-defined method of inducing autophagy. The present study suggests that SBA induces various degrees of autophagic cell death, followed by either necrosis or apoptosis at laters stage, depending on the cell type.

Photodynamic activity of BAM-SiPc, an unsymmetrical bisamino silicon(IV) phthalocyanine, in tumour-bearing nude mice.

BACKGROUND AND PURPOSE: Ever since the discovery of photodynamic therapy, there has been a continuous search for more potent photosensitizers. Towards that end, we have synthesized a number of novel phthalocyanine derivatives. The unsymmetrical bisamino silicon(IV) phthalocyanine BAM-SiPc is one of the most potent compounds. In in vitro cell culture, it exhibits high phototoxicity against a number of cancer cell lines. EXPERIMENTAL APPROACH: In the present investigation, the in vivo effect of BAM-SiPc was studied in the tumour-bearing nude mice model. The biodistribution of BAM-SiPc was followed to evaluate its tumour selectivity and rate of clearance. The tumour volume in the hepatocarcinoma HepG2- and the colorectal adenocarcinoma HT29-bearing nude mice was measured after photodynamic therapy. The level of intrinsic toxicity induced was also investigated. Finally, the metabolism of BAM-SiPc in the 'normal' WRL68 liver cells and the hepatocarcinoma HepG2 cells was compared. KEY RESULTS: The results not only showed significant tumour regression of HepG2 and growth inhibition of HT29 in the tumour-bearing nude mice, but also no apparent hepatic or cardiac injury with the protocol used. Histological analyses showed that apoptosis was induced in the solid tumour. BAM-SiPc could be metabolized by WRL68 liver cells but not by the hepatocarcinoma HepG2 cells. Unfortunately, BAM-SiPc did not show any specific targeting towards the tumour tissue. CONCLUSIONS AND IMPLICATIONS: The efficiency of BAM-SiPc in inhibiting tumour growth makes it a good candidate for further evaluation. Enhancement of its uptake in tumour tissue by conjugation with biomolecules is currently under investigation.

Room-temperature broadband emission of an InGaAs/GaAs quantum dots laser.

We report the first demonstration to our knowledge of an ultrabroad emission laser using InGaAs/GaAs quantum dots by cycled monolayer deposition. The device exhibits a lasing wavelength coverage of approximately 40 nm at an approximately 1160 nm center wavelength at room temperature. The broadband signature results from the superposition of quantized lasing states from highly inhomogeneous dots.

Peptidomics analysis of neuropeptides involved in copulatory behavior of the mollusk Lymnaea stagnalis.

Male copulation behavior in mollusks is controlled by an array of peptide messengers. In the present study, we have used a peptidomics approach employing liquid chromatography in conjunction with electrospray mass spectrometry to characterize peptides contained in the penial complex of the freshwater snail, Lymnaea stagnalis. In addition to the previously described peptides, we have identified a group of novel peptides that share the carboxyl termini of -FVRIamide. A cDNA cloning study revealed the organization of the precursor, which contains 20 peptide domains with the carboxyl termini of -F(X)RIamide which are flanked by many putative proteolytic sites including the KR and the less commonly occurring (G)K and (G)R sites. In addition, there are several monobasic R and dibasic RR and KK sites that may be used for processing. We then used MALDI-TOF/TOF-MS in a data-dependent mode, which selected all the molecular ion species with the predicted masses of the mature -F(X)RIamide peptides, and performed MS/MS analysis on these peptides. This approach allowed us to identify all the predicted -F(X)RIamide peptides. Immunocytochemistry showed the localization of -FVRIamide immunoreactive neurons in several central ganglia, and immunoreactive axons in the penial complex. Finally, application of synthetic -FVRIamide peptides to an in vitro posterior vas deferens preparation showed inhibitory effect on the spontaneous contraction/relaxation cycle of the vas deferens.

Stellettin A induces oxidative stress and apoptosis in HL-60 human leukemia and LNCaP prostate cancer cell lines.

The present study has demonstrated a differential cytotoxicity of stellettin A (1) between human leukemia HL-60 cells (IC50 0.4 microg/mL) and human prostate cancer LNCaP cells (IC50 120 microg/mL). Treatment of cells with 1 revealed the activation of NADPH oxidase, the dramatic generation of reactive oxygen species, and the dissipation of mitochondrial membrane potentials, with HL-60 cells being more sensitive than LNCaP cells by an order of magnitude. Immunoblotting analysis further demonstrated a stronger upregulation of the apoptosis marker proteins, FasL and caspase-3, in HL-60 cells, and pretreatment of cells with antisense oligonucleotide for caspase-3 abolished apoptosis. All available evidence suggests that 1 induces oxidative cell death through a FasL-caspase-3-apoptotic pathway.

Apoptotic activity of isomalabaricane triterpenes on human promyelocytic leukemia HL60 cells.

Four isomalabaricane triterpenes were isolated from marine sponge Geodia japonica [W.H. Zhang, C.T. Che, Isomalabaricane-type nortriterpenoids and other constituents of the marine sponge Geodia japonica, J. Nat. Prod. 64 (2001) 1489-1492. ] and their cytotoxicity was evaluated using a human promyelocytic leukemia HL60 cell line. Of the four triterpenes tested, geoditin A was the most cytotoxic to HL60 cells [IC50=3 microg/ml (<6.6 microM)], followed by stellettins A and B, whereas geoditin B exhibited relatively weak cytotoxicity. The treated cells manifested nuclear changes characteristic for apoptosis, and associated with dissipation of mitochondrial membrane potential, activation of caspase 3, and decrease of cytoplasmic proliferating cell nuclear antigen (PCNA), as demonstrated by fluorescence and immunofluorescence microscopy. When the HL60 cells were exposed to geoditin A ranging from 1.25 to 25 microg/ml, a dose-dependent increase of reactive oxygen species, a progressive dissipation of mitochondrial membrane potential, and an increase in annexin V-FITC binding were measured by flow cytometry. Taken together our results suggest that geoditin A markedly induced reactive oxygen species, decreased mitochondrial membrane potential and mediated a caspases 3 apoptosis pathway.

Tangutorine induces p21 expression and abnormal mitosis in human colon cancer HT-29 cells.

A novel beta-carboline alkaloid, tangutorine (benz[f]indolo[2,3-a]quinolizidine) was isolated from the leaves of Nitraria tangutorum L. [Duan JA, Williams ID, Che CT, Zhou RH, Zhao RH, Tangutorine: a novel beta-carboline alkaloid from Nitraria tangutorum. Tetrahedron Lett 1999;40:2593-6], and its unique structural characters led us to initiate a study of its potential anti-proliferation activity. The in vitro treatment with low doses of tangutorine slightly stimulated the proliferation of human colon cancer HT29 cells until at concentrations higher than 6.25 microg/ml when the cell numbers, cellular MTT reduction, and cell proliferation by 3H-thymidine incorporation decreased in a dose-dependent manner (IC50=15 microg/ml=48 microM). Morphological studies of cells by fluorescence and electron microscopy did not show features for apoptosis but only large vacuoles, swollen mitochondria and dense cytoskeletal filaments bunching in the cytoplasm. Immunoblotting analysis revealed a dramatic induction of cyclin kinase inhibitor p21 as well as an inhibition of topoisomerase II expression at 25 microg/ml tangutorine, thereby impeding cell progression from S to G2/M phase. Cells accumulated at G1 phase of the cell cycle at concentrations > or =50 microg/ml tangutorine. Interestingly, some cells escaped from prolonged growth arrest without cell division and resulted in binucleated and polyploid G1 cells. Taken all results together, tangutorine induced a p21 suppression of all cyclins and their associated kinases, such as the topoisomerase II, and thus inhibited normal DNA replication and mitosis.

The relationship between HPV16 and expression of CD44v6, nm23H1 in esophageal squamous cell carcinoma.

The esophageal squamous cell carcinoma (ESCC) has high incidence in Shaanxi Province of China. More and more researches indicated that human papillomavirus type 16 (HPV16) might play an important role in carcinogenesis of ESCC but the relationship between HPV16 and CD44v6, nm23H1 has not been elucidated. HPV16 was detected by amplifying HPV16 E6 gene through polymerase chain reaction (PCR) method and the expression of CD44v6, nm23H1 in 40 ESCCs and fifteen normal esophageal mucosa (NEM) from Shaanxi Province was examined by Streptavidin-Peroxidase (SP) method using monoclonal antibody specific to CD44v6 and nm23H1. The positive rates of HPV16 E6 gene, CD44v6 and nm23H1 were 60% (24/40), 65% (26/40) and 45% (18/40) respectively in ESCCs and 26.67% (4/15), 33.33% (5/15) and 86.67% (13/15) respectively in NEMs. There exited statistical difference for HPV16, CD44v6 and nm23H1 between NEMs and ESCCs respectively (p < 0.05). The relationship between HPV16 and the expression of CD44v6 in ESCCs was statistical significance (P = 0.021), but no significant correlation was found between HPV16 and the expression of nm23H1 (P = 0.436) in ESCCs. The infection rate of HPV16 had no statistical difference in all pathological features we observed, but the expression rates of CD44v6 and nm23H1 had statistical correlation with invasion (p = 0.001, 0.013) and lymph nodes metastasis (p = 0.014, 0.002) respectively. In different histology grade of ESSCs, the relationship between HPV16 and CD44v6 was statistical significance in grade I (p = 0.044) but was not in grade II (p = 0.165) and grade III (p = 0.658), however as to the expression of nm23H1 there exited no statistical significance in all histology grades of ESCC (p > 0.05). The expression rates of CD44v6 and nm23H1 were statistically different between grade I and II (p = 0.004, 0.016) respectively and between grade I and grade III (p = 0.014, 0.020), but not statistically different between grade II and III (p = 0.792, 0.943) respectively. Our data firstly suggested that there existed the statistical relationship between the infection of HPV16 and the expression of CD44v6 in ESCCs and that HPV16 may be involved in invasion and metastasis of ESCC.

Fas and activated caspase 8 in normal, Alzheimer and multiple infarct brains.

Using immunohistochemistry, Fas and caspase 8 positive sites were localized in the brains of Alzheimer patients and those with multiple infarcts as well as age-matched normal. The frontal, occipital and hippocampal regions were dissected out from these patients and normal individuals shortly after death. Positive Fas neurons were found in all these regions of normal individuals and Alzheimer patients, but not in the brains of patients with multiple infarct. Activated caspase 8 protein was found only in the brains of Alzheimer patients and not in the other groups. Plaques in the Alzheimer patients were positive for Fas and activated caspase 8. The former was localized in the membrane and the inside of the plaque whereas activated caspase 8 was only present in the interior of the plaque, as revealed by confocal microscopy. Our study shows that caspase 8 was activated in the Alzheimer brain and the presence of Fas and activated caspase 8 points to a possibility that at least some plaques were of intracellular origin as Fas was a cell membrane receptor and caspase 8 was a intracellular enzyme downstream to Fas. Furthermore, we had confirmed that in most neurons Fas and caspase 8 did not exist concomitantly and that the glial cells (astrocytes) did not express caspase 8 in Alzheimer patients.

Volvariella volvacea lectin activates mouse T lymphocytes by a calcium dependent pathway.

The immunomodulatory lectin, Volvariella volvacea lectin (VVL), isolated from the edible mushroom, Volvariella volvacea, has been shown to stimulate the expression of Th1 cytokines and the proliferative activity of mouse splenocytes (She et al. [1998]: Biochem Biophys Res Comm 247:106-111). In order to elucidate the mechanisms underlying these activities, we conducted a kinetic analysis of the early and late activation markers in mouse T lymphocytes: (1) flow cytometric analysis of calcium influx, (2) induction of activation molecules (CD25 and CD69), (3) expression and DNA-binding activity of the nuclear factor of activated T cells (NFAT), NFkappaB, and activation protein-1 (AP-1), (4) translational production of cytokines (interleukin-2 (IL-2) and interferon-gamma (IFNgamma)), and (5) cell proliferation by expression of proliferating cell nuclear antigen (PCNA) and tritiated thymidine incorporation. All results showed that VVL induced a rapid expression of CD69, CD25, NFAT, IL-2, and PCNA in a dose- and time-dependent manner, leading to lymphocyte proliferation. These effects brought about by VVL were more potent than those stimulated by equimolar concentrations of mitogenic lectin, concanavalin A (Con A). Cell activation and proliferation were mediated through a calcium-dependent pathway as demonstrated by a VVL-induced increase of intracellular calcium influx, and a proliferation inhibition by the Ca-dependent phosphatase calcineurin blocker-cyclosporin A (CsA). Taken all data together, VVL is a lectin which activates lymphocyte through successive calcium influx, nuclear localization of NFAT transcription factor, induction of activation markers, CD25 and CD69, intracellular cytokine production, and cell proliferation.

Fungal polysaccharopeptide inhibits tumor angiogenesis and tumor growth in mice.

Angiogenesis is crucial to tumor growth and metastasis, and interruption of this process is a prime avenue for therapeutic intervention of tumor proliferation. The present study has made use of the S180 tumor-bearing mouse model to investigate the polysaccharopeptide, PSP, isolated from the edible mushroom Coriolus versicolor, a herbal medicine known for its anti-angiogenesis properties. Quantitative analysis of microcorrosion casting of the tumor tissue showed more angiogenic features such as dense sinusoids and hot spots, in control (untreated) than in PSP-treated animals. Immunostaining of tumor tissues with antibody against the endothelial cell marker (Factor VIII) demonstrated a positive correlation in that both the vascular density and tumor weight were lower in mice treated with PSP. Morphometric analysis of corrosion casts revealed that, even though the total amount of new vessel production was reduced, the basic tumor type-specific vascular architecture was retained. However, the expression of vascular endothelial cell growth factor (VEGF) in these tumors was suppressed. In conclusion, anti-angiogenesis should be one of the pathways through which PSP mediated its anti-tumor activity.

Mitogenic activity of edible mushroom lectins.

A special group of lectins were isolated from three popular Asian edible mushrooms: Volvariella volvacea, Pleurotus flabellatus and Hericium erinacium, and their mitogenic activities towards mouse T cells were compared to the extensively investigated Agaricus bisporus lectin (ABL) and the Jack bean lectin, Concanavalin A (Con A). Among the four mushroom lectins tested, V. volvacea lectin (VVL) exhibited strong mitogenic activity as demonstrated by 3H-thymidine incorporation, which was at least 10-fold more effective than that of Con A, and the other mushroom lectins did not exhibit any proliferative activity. Treatment with VVL and ABL resulted in activation of the protein tyrosine kinase, p56lck, and expression of early activation markers, CD69 and CD25, but only VVL induced intracellular calcium influx while ABL triggered cell death. The calcium influx was sensitive to calcium channel antagonists such as nifedipine and verapamil. The P. flabellatus lectin (PFL) and H. erinacium lectin (HEL) did not stimulate p56lck expression and cell proliferation. Neither of these lectins interfered with Con A-mediated lymphocyte proliferation, which further indicated that both PFL and HEL were non-mitogenic. Taken all results together, VVL induced mitogenesis through T cell receptors and the subsequent calcium signaling pathway.

Wheat germ lectin induces G2/M arrest in mouse L929 fibroblasts.

Wheat germ lectin (WGA) is a cytotoxic lectin for many cell lines [Wang et al., 2000], but its underlying mechanism is not clear. In this report, we found that incubation of synchronized mouse L929 fibroblasts with WGA resulted in a dose-dependent reduction of intracellular incorporation of 3H-thymidine and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)-conversion activity (IC50 congruent with 0.4 microM). Fluorescein-conjugated WGA was demonstrated to transport from the cell surface into the paranuclear region of cultured L929 cells within 30 min, and subsequently evoked lipid peroxidation of plasma membrane and vacuolation in the cytoplasm of these cells. Studies with tritiated thymidine incorporation, immunofluorescence microscopy, immunoblotting analysis and flow cytometry revealed that WGA inhibited cell cycle progression after one replication, resulting in G2/M arrest and alteration of cell cycle regulatory proteins, particularly activation of p21Cip1/WAF1 and suppression of cyclin B and cdc 2. Although there was an increase of cytosolic caspase 3 and bax protein expression, no apoptotic bodies were observed by both fluorescence and transmission electron microscopy. These results suggest that WGA arrested L929 proliferation after one cell cycle in the G2/M phase through activation of the p21Cip1/WAF1 and suppression of Cyclin B-Cdc2.