Longitudinal changes in 18 F-THK5351 positron emission tomography in corticobasal syndrome.

BACKGROUND AND PURPOSE: Corticobasal syndrome (CBS) is pathologically characterized by tau deposits in neuronal and glial cells and by reactive astrogliosis. In several neurodegenerative disorders, 18 F-THK5351 has been observed to bind to reactive astrocytes expressing monoamine oxidase B. In this study, the aim was to investigate the progression of disease-related pathology in the brains of patients with CBS using positron emission tomography with 18 F-THK5351. METHODS: Baseline and 1-year follow-up imaging were acquired using magnetic resonance imaging and positron emission tomography with 18 F-THK5351 in 10 subjects: five patients with CBS and five age-matched normal controls (NCs). RESULTS: The 1-year follow-up scan images revealed that 18 F-THK5351 retention had significantly increased in the superior parietal gyrus of the patients with CBS compared with the NCs. The median increases in 18 F-THK5351 accumulation in the patients with CBS were 6.53% in the superior parietal gyrus, 4.34% in the precentral gyrus and 4.33% in the postcentral gyrus. In contrast, there was no significant increase in the regional 18 F-THK5351 retention in the NCs. CONCLUSIONS: Longitudinal increases in 18 F-THK5351 binding can be detected over a short interval in the cortical sites of patients with CBS. A monoamine oxidase B binding radiotracer could be useful in monitoring the progression of astrogliosis in CBS.

Brainstem manifestations in neuromyelitis optica: a multicenter study of 258 patients.

BACKGROUND: Neuromyelitis optica (NMO) is a severe autoimmune disease of the central nervous system characterized by spinal cord and optic nerve involvement. Brainstem manifestations have recently been described. OBJECTIVE: To evaluate the time of occurrence, the frequency and the characteristics of brainstem symptoms in a cohort of patients with NMO according to the ethnic background and the serologic status for anti-aquaporin-4 antibodies (AQP4-abs). METHODS: We performed a multicenter study of 258 patients with NMO according to the 2006 Wingerchuk criteria and we evaluated prospectively the frequency, the date of onset and the duration of various brainstem signs in this population. RESULTS: Brainstem signs were observed in 81 patients (31.4%). The most frequently observed signs were vomiting (33.1%), hiccups (22.3%), oculomotor dysfunction (19.8%), pruritus (12.4%), followed by hearing loss (2.5%), facial palsy (2.5%), vertigo or vestibular ataxia (1.7%), trigeminal neuralgia (2.5%) and other cranial nerve signs (3.3%). They were inaugural in 44 patients (54.3%). The prevalence was higher in the non-Caucasian population (36.6%) than in the Caucasian population (26%) (p<0.05) and was higher in AQP4-ab-seropositive patients (32.7%) than in seronegative patients (26%) (not significant). CONCLUSIONS: This study confirms the high frequency of brainstem symptoms in NMO with a majority of vomiting and hiccups. The prevalence of these manifestations was higher in the non Caucasian population.

Normal regulatory alpha/beta T cells effectively eliminate abnormally activated T cells lacking the interleukin 2 receptor beta in vivo.

Although interleukin 2 (IL-2) has been thought to be the most important cytokine for T cell growth, animals lacking IL-2 or a component of its receptor molecules have more expanded T cells with activated memory phenotype, indicating an indispensable role for the IL-2/IL-2 receptor system in regulating the size and activity of the T cell population. In this study, we investigated the possible mechanism of abnormal expansion of activated T cells in IL-2 receptor beta chain (IL-2Rbeta)(-/-) mice using the systems of bone marrow transplantation and T cell transfer. Here, we show that IL-2Rbeta(2/-) T cells in mice reconstituted with a mixture of IL-2Rbeta(2/-) and IL-2Rbeta(1/+) bone marrow cells did not develop into an abnormally activated stage, and that already activated IL-2Rbeta(2/-) T cells were effectively eliminated by IL-2Rbeta(1/+) T cells when both cells were cotransferred to T cell-deficient host mice. This regulation and/or elimination was dependent on T cells bearing alpha/beta type T cell receptor, especially on CD8(+) T cells and independent of the Fas-Fas ligand (FasL) system. IL-2Rbeta(1/+) T cells that eliminated activated IL-2Rbeta(2/-) T cells expressed FasL, perforin, granzyme B, and tumor necrosis factor alpha/beta. These results indicate a novel function of IL-2Rbeta that is necessary for the induction of regulatory T cells acting to eliminate activated T cells.

Treatment of MOG antibody associated disorders: results of an international survey.

INTRODUCTION: While monophasic and relapsing forms of myelin oligodendrocyte glycoprotein antibody associated disorders (MOGAD) are increasingly diagnosed world-wide, consensus on management is yet to be developed. OBJECTIVE: To survey the current global clinical practice of clinicians treating MOGAD. METHOD: Neurologists worldwide with expertise in treating MOGAD participated in an online survey (February-April 2019). RESULTS: Fifty-two responses were received (response rate 60.5%) from 86 invited experts, comprising adult (78.8%, 41/52) and paediatric (21.2%, 11/52) neurologists in 22 countries. All treat acute attacks with high dose corticosteroids. If recovery is incomplete, 71.2% (37/52) proceed next to plasma exchange (PE). 45.5% (5/11) of paediatric neurologists use IV immunoglobulin (IVIg) in preference to PE. Following an acute attack, 55.8% (29/52) of respondents typically continue corticosteroids for ≥ 3 months; though less commonly when treating children. After an index event, 60% (31/51) usually start steroid-sparing maintenance therapy (MT); after ≥ 2 attacks 92.3% (48/52) would start MT. Repeat MOG antibody status is used by 52.9% (27/51) to help decide on MT initiation. Commonly used first line MTs in adults are azathioprine (30.8%, 16/52), mycophenolate mofetil (25.0%, 13/52) and rituximab (17.3%, 9/52). In children, IVIg is the preferred first line MT (54.5%; 6/11). Treatment response is monitored by MRI (53.8%; 28/52), optical coherence tomography (23.1%; 12/52) and MOG antibody titres (36.5%; 19/52). Regardless of monitoring results, 25.0% (13/52) would not stop MT. CONCLUSION: Current treatment of MOGAD is highly variable, indicating a need for consensus-based treatment guidelines, while awaiting definitive clinical trials.

Intractable hiccup and nausea in neuromyelitis optica with anti-aquaporin-4 antibody: a herald of acute exacerbations.

BACKGROUND: Intractable hiccup and nausea (IHN) are unique symptoms in neuromyelitis optica (NMO). Recent studies have strongly suggested that the pathogenesis of NMO is closely associated with anti-aquaporin-4 (AQP4) antibody. However, clinical implications of IHN and the relationship with anti-AQP4 antibody remain unknown. METHODS: The past medical records of 35 patients with seropositivity for anti-AQP4 antibody were reviewed. We also followed the titres of anti-AQP4 antibody in a patient with NMO, who had newly developed IHN. RESULTS: Of the 35 patients, 15 patients (43%) had episodes of IHN. There was a total of 35 episodes of IHN in these 15 patients and, of the 35 episodes, hiccup was seen in 23 episodes (66%) and nausea was seen in 28 episodes (80%). The IHN frequently preceded (54%) or accompanied (29%) myelitis or optic neuritis. The IHN was often preceded by an episode of viral infection. The titres of anti-AQP4 antibody were remarkably increased when the intractable hiccup appeared in a case. CONCLUSIONS: IHN could be a clinical marker for the early phase of an exacerbation. Careful observation may be needed when INH is seen in patients with NMO, and the early initiation of the treatment could prevent subsequent neurological damage.

Preferential spinal central gray matter involvement in neuromyelitis optica. An MRI study.

OBJECTIVE: To delineate the MRI features that distinguish neuromyelitis optica (NMO) from multiple sclerosis (MS). METHODS: We compared the distribution of the spinal cord lesions by analyzing 1) lesion area, 2) lesion density (by superimposing the lesions onto the standard sections of the cervical and thoracic cord with appropriate transparencies using computer software), and 3) T1-hypointensity in axial sections of MRI in NMO and MS. RESULTS: In NMO, 60-70% of the cervical and thoracic cord MRI lesions occupied more than half of the cord area and mainly involved the central gray matter in the acute stage. In the chronic stage, half or more of the lesions were localized at the central gray matter region. The lesion superimposition analysis also revealed much higher densities in the central gray matter region than in the peripheral white matter regions. Two patients with NMO had T1-hypointense lesions in the central region. In contrast, over 80% of the lesions in MS were localized in the lateral and posterior white matter regions of the cord in the chronic as well as acute stage. Lesion densities were much higher in the lateral and posterior white matter regions than in the central gray matter region. None of the lesions in MS were T1-hypointense. CONCLUSIONS: These MRI findings strongly suggest a preferential involvement in the spinal central gray matter in NMO which is distinct from MS.

Loss of aquaporin 4 in lesions of neuromyelitis optica: distinction from multiple sclerosis.

Neuromyelitis optica (NMO) is an inflammatory and necrotizing disease clinically characterized by selective involvement of the optic nerves and spinal cord. There has been a long controversy as to whether NMO is a variant of multiple sclerosis (MS) or a distinct disease. Recently, an NMO-specific antibody (NMO-IgG) was found in the sera from patients with NMO, and its target antigen was identified as aquaporin 4 (AQP4) water channel protein, mainly expressed in astroglial foot processes. However, the pathogenetic role of the AQP4 in NMO remains unknown. We did an immunohistopathological study on the distribution of AQP4, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), activated complement C9neo and immunoglobulins in the spinal cord lesions and medulla oblongata of NMO (n = 12), MS (n = 6), brain and spinal infarction (n = 7) and normal control (n = 8). The most striking finding was that AQP4 immunoreactivity was lost in 60 out of a total of 67 acute and chronic NMO lesions (90%), but not in MS plaques. The extensive loss of AQP4 accompanied by decreased GFAP staining was evident, especially in the active perivascular lesions, where immunoglobulins and activated complements were deposited. Interestingly, in those NMO lesions, MBP-stained myelinated fibres were relatively preserved despite the loss of AQP4 and GFAP staining. The areas surrounding the lesions in NMO had enhanced expression of AQP4 and GFAP, which reflected reactive gliosis. In contrast, AQP4 immunoreactivity was well preserved and rather strongly stained in the demyelinating MS plaques, and infarcts were also stained for AQP4 from the very acute phase of necrosis to the chronic stage of astrogliosis. In normal controls, AQP4 was diffusely expressed in the entire tissue sections, but the staining in the spinal cord was stronger in the central grey matter than in the white matter. The present study demonstrated that the immunoreactivities of AQP4 and GFAP were consistently lost from the early stage of the lesions in NMO, notably in the perivascular regions with complement and immunoglobulin deposition. These features in NMO were distinct from those of MS and infarction as well as normal controls, and suggest that astrocytic impairment associated with the loss of AQP4 and humoral immunity may be important in the pathogenesis of NMO lesions.

Modulation of dendritic cell development by immunoglobulin G in control subjects and multiple sclerosis patients.

Intravenous immunoglobulin (IVIg) preparations are reportedly effective in inhibiting the relapse of multiple sclerosis (MS), but few reports have investigated the effect of IVIg on dendritic cells (DCs), which are thought to be involved in such relapses. In the system that uses monokines to differentiate DCs from peripheral blood monocytes (Mo-DCs), we investigated the effect of immunoglobulin G (IgG) on these antigen-presenting cells. Using monocytes derived from healthy volunteers, IgG partially inhibited the expression of CD1a, a marker of immature DCs (imDCs), and CD40 and CD80, which are markers associated with T cell activation. In contrast, IgG enhanced the expression of CD83, a marker of mature DCs (mDCs). Furthermore, IgG markedly inhibited the expression of CD49d [very late activation antigen (VLA)-4 alpha4-integrin], the adhesion molecule required for mDCs to cross the blood-brain barrier. We obtained similar results on all the aforementioned cell surface molecules investigated in both healthy controls and MS patients. In addition, IgG treatment of cells from both healthy controls and MS patients inhibited the production of interleukin (IL)-12, a cytokine associated with mDC differentiation, but did not inhibit the production of IL-10. These results suggested the possibility that IgG treatment, apart from its known ability to regulate inflammation, may help to prevent relapses of MS by controlling DC maturation, consequently inhibiting invasion of immune cells into the central nervous system and affecting the cytokine profile.

Ultraviolet light induces redox reaction-mediated dimerization and superactivation of oncogenic Ret tyrosine kinases.

The c-RET proto-oncogene encodes a receptor-type tyrosine kinase, and its mutations in the germ line are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B). Ret kinases are constitutively activated as a result of MEN2A mutations (Ret-MEN2A) or MEN2B mutations (Ret-MEN2B). Here we demonstrate that UV light (UV) irradiation induces superactivation of the constitutively activated Ret-MEN2A and Ret-MEN2B as well as activation of c-Ret. Before UV irradiation, small percentages of c-Ret (3-4%) and Ret-MEN2B (1-2%) and large percentages of Ret-MEN2A (30-40%) were dimerized through disulfide bonds. These dimerized Ret proteins were preferentially autophosphorylated, suggesting a close relation between up-regulated kinase activity and disulfide bond-mediated dimerization of Ret proteins. We found that UV irradiation promotes the disulfide bond-mediated dimerization of the Ret proteins, in close association with activation and superactivation of Ret kinases. UV irradiation also induced dimerization and activation of the extracellular domain-deleted mutant Ret (Ret-PTC-1). Interestingly, the levels of basic kinase activity and dimerization of Ret-PTC-1-C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-PTC-1 was replaced by alanine, were low and were not increased by UV irradiation. These results suggest that Ret-PTC-1 cysteine 376 is one of possibly multiple critical target amino acids of UV for Ret kinase activation. Overexpression of Cu/Zn superoxide dismutase in cells as a result of gene transfection prevented both the UV-mediated promotion of dimerization and the superactivation of Ret-MEN2A kinase. These results suggest that the UV-induced free radicals in cells attack intracellular domains of Ret to dimerize the kinase proteins for superactivation.

Clinical and MRI features of Japanese patients with multiple sclerosis positive for NMO-IgG.

This study investigates the relation between the serological status of NMO (neuromyelitis optica)-IgG and the clinical and MRI features in Japanese patients with multiple sclerosis. Serum NMO-IgG was tested in 35 Japanese patients diagnosed with multiple sclerosis, including 19 with the optic-spinal form of multiple sclerosis (OSMS), three with the spinal form of multiple sclerosis (SMS), and 13 with the conventional form of multiple sclerosis (CMS), which affects the brain. NMO-IgG was detected in 14 patients, 12 with OSMS and 2 with CMS. In these patients, longitudinally extensive (> 3 vertebral segments) spinal cord lesions (93% v 57%) and permanent, complete blindness (no perception of light) in at least one eye (50% v 0%) were the noticeable features as compared with NMO-IgG-negative OSMS. The two patients having CMS with NMO-IgG had unusual brain lesions, but in other respects had features suggesting OSMS. NMO-IgG was detected in more than half the number of patients with OSMS and in some patients with CMS. This newly discovered serum autoantibody was markedly associated with longitudinally extensive spinal cord lesions and with complete blindness, suggesting severe optic-spinal disease.

Induction of antitumor immunity in mice by allo-major histocompatibility complex class I gene transfectant with strong antigen expression.

An allo-major histocompatibility complex class I gene (H-2Kb) was transfected to murine mastocytoma P1.HTR (P815 subline) cells, after which several transfectant clones were obtained. Two clones, which expressed a low level of H-2Kb antigen, grew well and killed the syngeneic DBA/2 mice when they were inoculated ip. These mice lived longer than the mice given injections of the parental P1.HTR tumor. However, one clone, which expressed a high level of H-2Kb antigen, was rejected completely by the syngeneic DBA/2 mice and induced a generation of H-2Kb-specific cytotoxic T cells. Interestingly, the mice that had rejected the clone with high H-2Kb expression received strong anti-tumor immunity for rejection of the parental P1.HTR tumor challenged at the high dose.

Induction of high-grade tumor-specific immunity in a host using a cytotoxic T-lymphocyte clone specific for a stable tumor antigen on murine leukemia L1210.

T-cell clone K4L, the cell surface phenotypes of which were Thy-1+, Lyt-1-, Lyt-2+, and L3T4-, was established from the spleen cells of a murine leukemia L1210-immune mouse. Clone K4L was specific for antigen B on L1210, and this antigen was different from antigen A for which the previously reported T-cell clone K7L was specific. K4L possessed cytotoxicity and tumor growth-inhibitory activity against both L1210 and antigen A loss variant, L1210-K7L-, but not against syngeneic tumor P388 or L5178Y. Previously we showed that antigen A was lost frequently for generation of antigen loss variants. In contrast, antigen B was barely found to be lost. When mice were inoculated with L1210 plus a moderate dose of K4L, the tumor grew after initial suppression but this newly emerging tumor was K4L sensitive and was ultimately rejected. The mice initially given L1210 plus K4L attained a high-grade tumor-specific immunity for rejecting the subsequently challenged high-dose (10(7) cells) L1210. This immunity did not involve any bystander antitumor activity against the third party P388 lymphoma that was injected together with L1210 but accompanied the increase in the L1210-specific cytotoxic T-lymphocyte activity. Evidence was provided that the live L1210, the outgrowth of which was inhibited by K4L, induced an effective immune response of radiation-sensitive host lymphocytes including L3T4+ helper T-cells. Taken together, our results show a novel strategy for inducing high-grade host-dependent antitumor immunity by use of a cytotoxic T-lymphocyte clone specific for a stable tumor-specific transplantation antigen.

Dynamics of generation of antigen loss variants from L1210 murine leukemia clones detected by a tumor-specific T-cell clone.

Originally T-cell clone K7L-sensitive L1210 murine leukemia clones were tested for their capacity to generate K7L-insensitive variants at various times after cloning. All of the L1210 clones (L1210/1, -2, -4, and -7) maintained in vitro for 1 month were severely inhibited in their growth in the culture in which K7L was added and in mice given injections of K7L at the initial stage. This indicated that any L1210 clone tested was not a mixture of K7L-sensitive and K7L-insensitive clones at the time of cloning. By both in vivo and in vitro K7L-mediated tumor suppression assays, K7L-insensitive antigen loss variants were then found to be generated from some (L1210/4, L1210/7) but not other (L1210/1, L1210/2) originally K7L-sensitive L1210 clones during 1 month of maintenance. Ratios of variant cells to total clone cells 1 month after cloning were estimated around 0.1% for L1210/7, 0.01% for L1210/4, and less than 0.001% (undetectable) for L1210/1 and L1210/2. Neither L1210/1 nor L1210/2 generated detectable K7L-insensitive variant cells during long-term (14-month) maintenance. All of the ten subclones of L1210/7 which were obtained 7 or 11 months after the initial cloning of L1210/7 were K7L sensitive, and not all the subclones generated K7L-insensitive variants in 1-2 months of maintenance after recloning. However, all of the subclones of L1210/7 which were maintained for 7 months generated antigen loss variants. All eight clone cells obtained from original L1210 and K7L-insensitive L1210 expressed H-2Kd and H-2Dd antigens detected by H-2Kd or Dd-specific cytotoxic T-lymphocyte clones or monoclonal antibodies. These results suggest that the antigen loss variants arise in originally K7L-sensitive L1210 clones at different times after cloning, and the probability of generation of the variants is clonally determined. The antigen loss variants seem to be generated by rare (once per 1 to 2 months or less frequent) chance with unproportionally rapid growth rather than by more frequent development for simple accumulation. The ratio of K7L-insensitive variant cells to total L1210/7 cells did not increase progressively during long-term (13 months or more) maintenance in vivo or in vitro and was always below 0.1%. It was suggested that the population size of antigen loss variants was controlled biphasically.

Identification and characterization of a unique tumor-associated surface antigen on L1210 leukemia cells recognized by semisyngeneic antisera.

The tumor-associated surface antigen on L1210 leukemia cells was studied by immunofluorescence staining and immunoprecipitation. Anti-L1210 serum was prepared in BALB/c X DBA/2 F1 mice by priming with a hybrid of L1210 and human Lesch-Nyhan fibroblast cells and hyperimmunizing with L1210 leukemia cells. This hyperimmune serum was able to demonstrate specific surface fluorescence on L1210 cells, while the antiserum did not react with various mouse tumor cell lines, normal lymphoid tissues, or mitogen-activated lymphoid cells. The anti-L1210 serum immunoprecipitated a single polypeptide with a molecular weight of 90,000 from 125I-labeled L1210 cells. The expression of this antigen was enhanced by tumor-promoting agent and heat shock treatment. The biological significance of the L1210-specific cell surface antigen is discussed.

Complement-dependent and -independent aquaporin 4-antibody-mediated cytotoxicity in human astrocytes: Pathogenetic implications in neuromyelitis optica.

BACKGROUND: Neuromyelitis optica (NMO) is an inflammatory disease caused by the aquaporin (AQP)-4-antibody. Pathological studies on NMO have revealed extensive astrocytic damage, as evidenced by the loss of AQP4 and glial fibrillary acidic protein (GFAP), specifically in perivascular regions with immunoglobulin and complement depositions, although other pathological patterns, such as a loss of AQP4 without astrocyte destruction and clasmatodendrosis, have also been observed. Previous studies have shown that complement-dependent antibody-mediated astrocyte lysis is likely a major pathomechanism in NMO. However, there are also data to suggest antibody-mediated astrocyte dysfunction in the absence of complement. Thus, the importance of complement inhibitory proteins in complement-dependent AQP4-antibody-mediated astrocyte lysis in NMO is unclear. In most of the previous studies, the complement and target cells (astrocytes or AQP4-transfected cells) were derived from different species; however, the complement inhibitory proteins that are expressed on the cell surface cannot protect themselves against complement-dependent cytolysis unless the complements and complement inhibitory proteins are from the same species. To resolve these issues, we studied human astrocytes in primary culture treated with AQP4-antibody in the presence or absence of human complement and examined the effect of complement inhibitory proteins using small interfering RNA (siRNA). METHODS: Purified IgG (10 mg/mL) was obtained from 5 patients with AQP4-antibody-positive NMO, 3 patients with multiple sclerosis (MS), and 3 healthy controls. Confluent human astrocytes transfected with Venus-M1-AQP4-cDNA were incubated with IgG (5% volume). After washing, we cultured the cells with human complements with or without heat inactivation. We observed time-lapse morphological and immunohistochemical changes using a fluorescence microscope. We also evaluated cytotoxicity using a propidium iodide (PI) kit and the lactate dehydrogenase (LDH) assay. RESULT: AQP4-antibody alone caused clustering and degradation followed by endocytosis of membraneous AQP4, thereby resulting in decreased cellular adherence and the shrinkage of astrocytic processes. However, these changes were partially reversed by the removal of IgG in culture. In contrast, following the application of AQP4-antibody and non-heated human complements, the cell bodies and nuclei started to swell. At 3 h, most of the astrocytes had lost mobility and adherence and were eventually destroyed or had swollen and were then destroyed. In addition, the remaining adherent cells were mostly PI-positive, indicating necrosis. Astrocyte lysis caused by rabbit complement occurred much faster than did cell lysis with human complement. However, the cell lysis was significantly enhanced by the transfection of astrocytes with siRNA against human CD55 and CD59, which are major complement inhibitory proteins on the astrocyte membrane. AQP4-antibody-negative IgG in MS or control did not induce such changes. CONCLUSION: Taken together, these findings suggest that both complement-dependent and complement-independent AQP4-antibody-mediated astrocytopathies may operate in NMO, potentially contributing to diverse pathological patterns. Our results also suggest that the effect of complement inhibitory proteins should be considered when evaluating AQP4-antibody-mediated cytotoxicity in AQP4-expressing cells.

cDNA cloning of mouse ret proto-oncogene and its sequence similarity to the cadherin superfamily.

We report the nucleotide sequence of the mouse ret proto-oncogene (proto-ret) and the deduced amino acid sequence. It encodes a transmembrane tyrosine kinase of 1115 amino acids that shows 83% homology with the human proto-Ret protein. The amino acid sequence revealed that the structures of the extracellular domain as well as the tyrosine kinase domain are similar in human and mouse proto-Ret proteins. Interestingly, the extracellular domains of both human and mouse proto-Ret proteins contain a cadherin-related sequence that is known to be important for Ca(2+)-dependent homophilic binding of the cadherins. When we examined transcription of the proto-ret gene in a variety of mouse tissues, it was detected in lymph nodes of C3H/HeJ-gld/gld mice and in normal mouse spinal cord. Furthermore, its transcription was found in the Neuro-2a mouse neuroblastoma cell line but not in 13 other rodent cell lines surveyed. Western blot analysis showed that proto-Ret proteins are expressed as 140-kDa and 160-kDa glycoproteins in Neuro-2a cells.

Clustered cysteine residues in the kinase domain of v-Src: critical role for protein stability, cell transformation and sensitivity to herbimycin A.

We have previously reported the activation of Src by mercuric chloride based on the sulfhydryl modification. To evaluate the significance of cysteine residues in v-Src, we replaced each cysteine to alanine by oligonucleotide-directed mutagenesis and examined its effect on cell transformation. Of ten cysteine residues scattered over v-Src, four cysteines clustered in kinase domain, Cys483, Cys487, Cys496 and Cys498, were important for protein stability and cell transformation, whereas those in SH2 domain were dispensable. A single mutation in Cys498 yielded suppression of kinase activity and a temperature-sensitivity in anchorage independent growth. Double mutation either in Cys483/Cys487 or in Cys496/Cys498 yielded clear temperature-sensitivity in cell transformation and in stability of Src protein. Instability of Src protein was magnified by quadruple mutation in the cysteines, which decreased the half-life of Src to be less than one quarter of that of wild-type. In addition, both Cys483/Cyr487 and Cys496/Cys498 kinases became resistant to in vitro inactivation by herbimycin A, which directly inactivates v-Src in addition to its effect on HSP90. Taken together, our results strongly suggest that the cysteine clustered motif of v-Src are critical for protein stability, cell transformation and in vitro inactivation by herbimycin A.

Establishment and characterization of a malignant melanocytic tumor cell line expressing the ret oncogene.

We established a cell line (designated Mel-ret) from a melanocytic tumor developed in a metallothionein/ret transgenic mouse. Unlike primary melanocytic tumors, which did not show malignant features, when the Mel-ret cells were transplanted into nude mice they invaded into surrounding tissues and had metastatic ability. Although the Ret proteins were expressed at similar levels in the cell line and the primary tumors, the level of tyrosine phosphorylation in the Mel-ret cells was much higher than that in the primary tumors. In particular, an 85-kDa tyrosine-phosphorylated band was specifically detected in the Mel-ret cells. These results suggest that the increase in tyrosine phosphorylation may be responsible for malignant transformation of the Mel-ret cells. Immunofluorescence and cell fractionation studies showed that the Ret proteins and most of tyrosine-phosphorylated proteins in the Mel-ret cells localized in the membrane fraction. No activation of phosphatidyl-inositol-3 kinase (PI-3 kinase), a target protein for several tyrosine kinases, was detected in the Mel-ret cells.

Linkage between melanocytic tumor development and early burst of Ret protein expression for tolerance induction in metallothionein-I/ret transgenic mouse lines.

We examined the basis of the all or none difference in inducing melanocytic tumor development among three transgenic mouse lines (304, 192 and 242) to which the same promoter-enhancer (metallothionein-I) and oncogene (ret) were introduced. We initially demonstrated that both skin melanosis and Ret protein expression in skin, thymus and brain first became detectable before or immediately after birth in the mice of the tumor developing lines (304 and 192), whereas they became detectable a few days after birth in the mice of the non-tumor developing line (242) by Western blotting and immunohistochemical analysis. Interestingly, the Ret protein expression in skin developed rapidly after birth as a burst with peak levels on 0.5-1.5 day newborns of lines 304 and 192 and on 7.0-7.5 day-old mice of line 242. The levels of autophosphorylation of Ret kinase in skin were, however, invariable among these three transgenic mouse lines. The mice of line 242, but not those of lines 192 and 304, responded to Ret protein immunization by increased antigen-dependent lymphocyte proliferation and T-cell-mediated tumor growth suppression in vitro. Furthermore, ret-transgenic mice of line 242, but not line 304, rejected the subcutaneously transplanted tumors that had originally developed in a mouse of line 304. These results suggest that whether oncogene product-specific-tolerance is established or not to antitumor immunity may be decided by the dynamics of ret oncogene expression before and after delivery and this is the primary factor determining development or non-development of melanoma.