Signalling during hypoxia in human T lymphocytes – critical role of the src protein tyrosine kinase p56Lck in the O2 sensitivity of Kv1.3 channels

T lymphocytes encounter hypoxia when they migrate to pathological sites such as tumours and wounds. The inability of T cells to provide an efficient defence at these sites can in part be explained by the hypoxic environment. Kv1.3 channels, important components of the T cell activation process are inhibited by hypoxia and their inhibition accounts for a hypoxia-induced decrease in T cell proliferation. Although Kv1.3 channels play a key role in T cell O₂ sensing, the signalling mechanisms mediating their response to hypoxia are still not understood. In this study, we show that the src-protein tyrosine kinase p56Lck (Lck) is required for Kv1.3 channel response to hypoxia. Pre-exposure to the src inhibitor PP2 abolished the hypoxia-induced inhibition of Kv1.3 channels in primary human T lymphocytes. Moreover, Kv1.3 channel sensitivity to hypoxia was lost in Lck-deficient Jurkat T cells. Further studies with recombinant Kv1.3 channels showed that Kv1.3 channels lack intrinsic O₂ sensitivity, but delivery of Lck into the cells and transfection of a constitutively active Lck (Y505FLck) restored their sensitivity to hypoxia. Although Lck is necessary for the Kv1.3 channel response to hypoxia, it does not directly inhibit Kv1.3 channels. Indeed, under normal oxygen tension, delivery of active Lck into L929 cells and overexpression of Y505FLck did not decrease recombinant Kv1.3 currents. On the contrary, activation of endogenous src kinases increased wild-type Kv1.3 currents in T lymphocytes. Our findings indicate that Lck is required for the acute response to hypoxia of human T lymphocytes as it is necessary to confer O₂ sensitivity on Kv1.3 channels.     

Increased production of lipocalin-type prostaglandin D synthase in leptomeningeal cells through contact with astrocytes

Lipocalin-type prostaglandin D synthase (L-PGDS) is dominantly expressed in the leptomeninges surrounding the brain and secreted into the cerebrospinal fluid as β-trace, a major cerebrospinal fluid protein. To examine the interaction between the leptomeninges and the brain parenchyma, we co-cultured rat leptomeningeal cells with cells dissociated from the neonatal rat cortex and found that the production of L-PGDS was remarkably increased after the co-cultivation. A similar increase in L-PGDS production was observed by the co-culturing of the leptomeningeal cells with cells dissociated from astrocyte-rich cultures or with 1321-N1 astrocytoma cells. When a crude membrane fraction prepared from 1321-N1 cells was added to leptomeningeal cell cultures, L-PGDS gene expression was slowly increased up to 48 h after the addition. These results indicate that leptomeningeal cells enhance their L-PGDS production by a slow activation of L-PGDS gene expression through their contact with astrocytes.     

Analgesic action of loperamide,an opioid agonist,and its blocking action on voltage-dependent Ca2+ channels

We investigated the relationship between the antinociceptive effect of the opiate agonist loperamide at the spinal level and its inhibitory effect on calcium influx. Intrathecal administration of loperamide showed a significant antinociceptive effect in the formalin test, which was not prevented by naloxone. On the other hand, no significant effects were observed by nicardipine, an L-type specific blocker, or by BAY K8644, an L-type specific agonist, suggesting no significant role of L-type calcium channels in nociceptive signal transduction. Loperamide suppressed the calcium influx in dorsal root ganglion neurons. As the antinociceptive effect of loperamide was not affected by naloxone or other calcium channel blocking toxins, and loperamide showed a direct inhibitory effect on calcium-influx, the analgesic effect of intrathecally injected loperamide might be due to its blockade of the voltage-dependent calcium channels at the terminals of the primary afferent fibers.     

A new pseudo-peptide of Arg-Gly-Asp (RGD) with inhibitory effect on tumor metastasis and enzymatic degradation of extracellular matrix

A series of pseudo-peptide analogs of the Arg-Gly-Asp (RGD) sequence of fibronectin have been synthe-sized, and their anti-metastatic effects in mice and inhibitory effects on tumor cell invasion in vitro have been examined. The partially modified retro pseudo-peptide of RGD, R_rev-COCH₂CO-D (FC-63), was more effective in inhibiting tumor metastasis than the original RGDS peptide. Replacement of the malonyl moiety of FC-63 with a carboxyethylene linkage (R_rev-COCH₂CH₂-D, FC-303 ) achieved more potent inhibition of lung metastasis of melanoma cells than FC-63. Among the analogs, FC-336, a p-xylylendiamine derivative having two FC-303 moieties, showed the most potent inhibitory effect on experimental lung metastasis produced by i.v. co-injection with B16-BL6 melanoma or colon 26 M3.1 cells in a dose-dependent manner. Multiple administrations of FC-336 after tumor inoculation also showed efficient therapeutic potency against spontaneous lung metastasis of B16-BL6 melanoma in mice. Furthermore, FC-336 effectively inhibited the invasion, migration and adhesion of tumor cells in vitro, but its inhibitory effects were not more than those of RGDS peptide. Zymography analysis revealed that FC-336 inhibited the degradation of gelatin substrate by matrix metalloproteinases (MMPs) produced by tumor cells, while the RGDS peptide did not affect the enzymatic degradation. These findings indicate that the pseudo-peptides of the RGD sequence, possessing the inhibitory property of the degradation by MMPs differently from original RGD-containing peptides, may be advantageous and useful in preventing tumor metastasis. © Rapid Science 1998     

Requirement of IL-5 for induction of autoimmune hemolytic anemia in anti-red blood cell autoantibody transgenic mice

IL-5, IL-10 and lipopolysaccharide (LPS) are known to activateB-1 cells in vivo in normal mice and anti-red blood cell autoantibodytransgenic mice (HL mice). To assess the exact role of IL-5in proliferation and activation of peritoneal B-1 cells, weanalyzed IL-5 receptor chain-deficient HL (IL-5R^–/–x HL) mice generated by the cross between IL-5R^–/–and HL mice. In IL-5R^–/– x HL mice, Ig-producingB-1 cells in the peritoneal cavity were negligible, althoughthe total number of B-1 cells in the peritoneal cavity wereas many as 30% of that in HL mice. Moreover, LPS- or IL-10-induceddifferentiation of B-1 cells into antibody-producing cells wasseverely impaired in IL-5R^–/– x HL mice. We alsoused in vivo 5-bromo-2'-deoxyuridine labeling to estimate theproliferation of B-1 cells in IL-5R^–/– mice. Theabsence of IL-5R did not affect spontaneous proliferation ofperitoneal B-1 cells. However, induced proliferation of peritorealB-1 cells by oral administration of LPS was markedly impairedin IL-5R^–/– mice. These results suggest that IL-5is required for activation-associated proliferation of B-1 cellsbut not for their spontaneous proliferation and support theidea that IL-5 plays an important role on the induction of autoantibodyproduction from B-1 cells.     

Detection of the Epstein-Barr virus in primary gastric lymphoma by in situ hybridization

The Epstein-Barr virus (EBV) has been shown to be associated with numerous human malignancies including Burktt's lymphoma and nasopharyngeal lymphoepithelioma. In addition, some typical gastric adenocarcinomas were also recently reported to demonstrate EBV relevance. The present study was designed to detect EBV in primary gastric lymphoma, using the in situ hybridization (ISH) method, in which oligo-nucleotide probes for the EBERl RNA and the EBV DNA W region have been used. Of the 49 cases of primary gastric lymphoma studied, which all showed B cell immunopheno-type, EBER1 sequences could only be found in four cases, including two low-grade cases and two high-grade cases of histological subtypes while the number of positive cells was less than 50% of the tumor cells. In one case of low-grade mucosa associated lymphoid tissue (MALT) lymphoma, the EBER1 -positive neoplastic cells were found in the regional lymph node, but the primary site of the stomach showed no positive signals. The EBV presence was further confirmed by the EBV DNA ISH. Using the ISH method, rare or occasional positive lymphoid cells (probably non-tumorous bystander cells) could be detected in 10 other cases including all histological subtypes. The present study shows that only a small proportion of primary gastric lymphoma is associated with EBV, and such positive cases could be found in both high- and low-grade histological subtypes. It is also suggested that the EBV presence in the neoplastic cells of some cases of primary gastric lymphoma is most likely a secondary phenomenon.     

Analysis of acute-phase toxicities of intensity-modulated proton therapy using a model-based approach in pharyngeal cancer patients

Pharyngeal cancer patients treated with intensity-modulated proton therapy (IMPT) using a model-based approach were retrospectively reviewed, and acute toxicities were analyzed. From June 2016 to March 2019, 15 pharyngeal (7 naso-, 5 oro- and 3 hypo-pharyngeal) cancer patients received IMPT with robust optimization. Simulation plans for IMPT and intensity-modulated X-ray therapy (IMXT) were generated before treatment. We also reviewed 127 pharyngeal cancer patients with IMXT in the same treatment period. In the simulation planning comparison, all of the normal-tissue complication probability values for dysphagia, dysgeusia, tube-feeding dependence and xerostomia were lower for IMPT than for IMXT in the 15 patients. After completing IMPT, 13 patients completed the evaluation, and 12 of these patients had a complete response. The proportions of patients who experienced grade 2 or worse acute toxicities in the IMPT and IMXT cohorts were 21.4 and 56.5% for dysphagia (P < 0.05), 46.7 and 76.3% for dysgeusia (P < 0.05), 73.3 and 62.8% for xerostomia (P = 0.43), 73.3 and 90.6% for mucositis (P = 0.08) and 66.7 and 76.4% for dermatitis (P = 0.42), respectively. Multivariate analysis revealed that IMPT was independently associated with a lower rate of grade 2 or worse dysphagia and dysgeusia. After propensity score matching, 12 pairs of IMPT and IMXT patients were selected. Dysphagia was also statistically lower in IMPT than in IMXT (P < 0.05). IMPT using a model-based approach may have clinical benefits for acute dysphagia.     

Angiosarcoma of the breast: Report of a case and autopsy findings

We report a case of angiosarcoma of the breast and the autopsy findings. The patient was a 35-year-old premenopausal woman who complained of a tumor in her left breast. We found a tumor measuring 55 mm in diameter in the lower external quadrant. The tumor was elastic and soft, smooth surfaced, well-defined and mobile. Dimpling sign or change of skin color were not observed. Clinically it was diagnosed as phyllodes tumor, but tumorectomy revealed primary angiosarcoma of the breast. Further extended surgery was recommended, but the patient refused additional therapy. Histological findings revealed a free surgical margin and neither lymph node metastasis nor distant metastasis were clinically observed. Seven months later, local recurrence in the same breast was recognized and finally radical mastectomy was carried out. Histological findings showed recurrence of angiosarcoma in the left breast but lymph node metastasis was not detected. Two months after mastectomy, metastases to the cervical and thoracic vertebrae were observed and radiation therapy was performed. Sixteen months from onset, she died due to multi-organ failure as general metastases of angiosarcoma. At autopsy, metastases to many organs including the digestive system were observed. The incidence of primary angiosarcoma of the breast is low but its prognosis is poor. This case emphasized the difficulties in clinical diagnosis and treatment for the angiosarcoma of the breast.     

Low grade amplification of MDM2 gene in a subset of human breast cancers without p53 alterations

MDM2 protein is thought to bind to p53 tumor suppressor protein leading to inhibition of p53-mediated transactivation. Amplification of the MDM2 gene has been frequently observed in human sarcoma, and relevant overexpression of the MDM2 protein is assumed to contribute to tumorigenesis through inactivation of the p53 function. In order to determine whether MDM2 amplification plays a role in the development of human breast cancer without genetic alteration of p53, we analyzed, MDM2 gene amplification by quantitative hybridization and genetic alteration of p53, in 32 primary tumors and 26 metastatic lymph nodes. Low grade amplification of the MDM2 gene (2-6 fold) was observed in four cases, none of which showed even subtle genetic alterations of p53 or loss of alleles on 17p. Moreover, in three of the four cases with MDM2 gene amplification, the level of gene amplification in the metastatic lymph nodes was slightly higher than that in the primary tumors. These results, taken together with previous findings, suggest that a subset of breast cancers without genetic alteration of p53 may also arise by inactivation of the p53 function through interaction with the overexpressed MDM2 protein induced by gene amplification.     

Association of Epidermal Growth Factor-related Peptides and Type I Receptor Tyrosine Kinase Receptors with Prognosis of Human Colorectal Carcinomas

The frequency of expression and localization of cripto-1 (CR-1),amphiregulin (AR), transforming growth factor alpha (TGF), epidermalgrowth factor receptor (EGFR) and erbB-2 were examined by immunohistochemistryin 45 carcinomas and adjacent non-involved normal colon mucosa.Thirty (66.7%), 24 (53.3%), 23 (51.1%), 23 (51.1%) and 13 (28.9%)of the 45 carcinomas showed positive staining for CR-1, AR,TGF, EGFR and erbB-2, respectively, whereas 7 (15.5%), 17 (37.7%),15 (33.3%), 20 (44.4%) and 0 (0%) of the corresponding non-involvednormal mucosa specimens were reactive. Among 13 carcinomas withlymph node involvement, 10 (76.9%), 8 (61.5%), 10 (76.9%), 8(61.5%) and 7 (53.8%) exhibited positive staining for CR-1,AR, TGF-, EGFR and erbB-2, respectively. There was a statisticallysignificant association between the frequency of either TGF(P<0.05) or erbB-2 (P<0.05) expression and lymph nodemetastasis. In addition, a signficantly higher frequency ofpositive staining for TGF was observedin Dukes' grade C carcinomas(P<0.05). Finally, significant trends for coexpression ofEGFR and either TGF (P<0.01) or AR (P<0.05) were detectedin carcinomas. These data suggest that AR and TGF may play animportant role in the development of colorectal carcinomas throughan autocrine mechanism involving EGFR, and demonstrate thatTGF and erbB-2 may be more reliable indicators of metastasisor prognosis than CR-1, AR or EGFR in human colon cancers.