Tsutomu Narita

The Autocrine Loop of Epidermal Growth Factor Receptor‐Epidermal Growth Factor/Transforming Growth Factor‐α in Malignant Rhabdoid Tumor Cell Lines: Heterogeneity of Autocrine Mechanism in TTC549

To investigate the effects of the autocrine loop of epidermal growth factor receptor (EGFR)‐epidermal growth factor (EGF)/transforming growth factor‐α (TGF‐α) on the proliferation and differentiation of malignant rhabdoid tumor (MRT), we used five MRT cell lines, TM87‐16, STM91‐01, TTC549, TTC642, and YAM‐RTK1. RT‐PCR analyses revealed expression of EGFR mRNA in all MRT cell lines. In contrast, the expression of either EGF or TGF‐α mRNA was detected in all MRT cell lines. Expression of EGF, TGF‐α, and EGFR as determined by immunocytochemical staining and in situ hybridization, correlated with the results of RT‐PCR. Upon differentiation‐induction with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), in TTC549, showing an expression of TGF‐α but not EGF initially, de novo expression of EGF mRNA appeared abruptly on day 2 of TPA treatment. To confirm the EGFR‐EGF/TGF‐α autocrine loop, we used TGF‐α, EGF, and their antibodies in the cultures. Monoclonal antibody (mAb) to EGFR alone significantly inhibited the growth of cell line TTC549. However, mAb to EGF or TGF‐α could inhibit proliferation of this cell line only when administrated together. Our findings would suggest that growth of the TTC549 cell line is constitutionally regulated by TGF‐α/EGFR, but that inhibition of this autocrine mechanism results in transient activation of an autocrine loop involving EGF/EGFR. Our results may indicate the presence of two different autocrine loops of EGFR‐EGF and/or EGFR‐TGF‐α in MRT cell lines. The heterogeneity of autocrine mechanisms found in MRT cell lines would be consistent with the multiphenotypic diversity and aggressive characteristics of this enigmatic tumor.

Neuronal Apoptosis Mediated by IL-1β Expression in Viral Encephalitis Caused by a Neuroadapted Strain of the Mumps Virus (Kilham Strain) in Hamsters

The neuroadapted Kilham strain of the mumps virus produces lethal encephalitis in newborn hamsters after intracerebral inoculation. The pathogenesis of this encephalitis is not fully understood, but recently, apoptosis and associated cytokine production have been recognized to be major pathologic mechanisms by which viruses cause injury to neuronal host cells. To analyze the main factors producing brain injury in this viral encephalitis, the following questions were investigated: (1) does the virus induce neuronal apoptosis and (2) does expression of cytokines regulate the induction of neuronal apoptosis? Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was used as a marker of neuronal apoptosis and TUNEL-positive neurons were widespread in the infected cerebral cortex. DNA fragmentation yielding DNA ladders characteristic of apoptosis was also observed in infected hamster brain tissue. Apoptotic cells in infected brains were observed after the appearance of inflammatory changes. Overexpression of IL-1 beta, but not TNF-alpha or Fas-L, was clearly detected in infected brains, as determined by Western blot and RT-PCR. Immunohistochemistry revealed a striking correlation between IL-1 beta expression and neuronal apoptosis. Injection of recombinant IL-1 beta into normal hamster brain resulted in neuronal apoptosis in cerebral cortex. On the other hand, neutralizing IL-1 beta antibodies decreased the number of cells undergoing apoptosis in infected hamster brains and subsequent death. We conclude that the fatal encephalitis induced by the Kilham strain of the mumps virus is mediated by immunopathological processes and that overexpression of IL-1 beta, which mediates the induction of neuronal apoptosis, may play a major role in these processes.

A patient with Kawasaki disease who developed acute urinary retention due to pelvic neuroblastoma.

Neuroblastoma is one of the common solid tumours in childhood. Kawasaki disease (KD) is not an uncommon acute febrile illness in Japan. There have been only two reports of KD patients having neuroblastoma [1,2]. Nonetheless, there is no reported case of acute KD developing urinary retention due to neuroblastoma. A 4-month-old boy was admitted to our hospital because of KD. He had a fever and skin rash for more than 5 days. Hyperemia of the eyes, oral cavity and tongue, left cervical lymph node swelling (diameter 1 cm) and finger scleroedemas were also observed. CRP was 15.7 mg/dl and blood culture was negative. Three days after admission, he suddenly developed acute urinary retention with urinary bladder distension and a decrease in urinary output. A pelvic echogram and an abdominal CT scan showed a small solid mass behind the bladder. Subsequent determination of urinary vanillylmandelic acid and homovanillic acid excretion revealed elevated levels. Surgical resection of the mass was successful. The histopathological finding on the resected tumour was a stroma poor, undifferentiating neuroblastoma according to the Shimada classification. Swelling of vascular endothelial cells and thromboses with perivascular leucocytic infiltration were observed in the resected tumour specimens. The thrombocyte count was elevated to 74.5·10/ll on the 11th day. He was treated with acetylsalicylic acid and he has been free from the disease for more than 9 years. In addition, no complication of coronary disease was noted. Neuroblastoma complicating KD is extremely rare. The incidence of neuroblastoma in Japan is 12–18/ 100,000 among children of 0 to 4 years of age, while that of KD is about 222/100,000. Previously, Yanagisawa et al. [3] reported a 6-month-old male infant who died of KD and had a small encapsulated mass in the left paravertebral region (T–10) found at necropsy. The histopathological diagnosis was neuroblastoma although he had no clinical signs of neuroblastoma. There was no histopathological documentation about an effect of KD on neuroblastoma. A further KD patient developed a neuroblastoma a few years later [2]. In addition, there have been no reports of symptomatic neuroblastoma complicating an acute phase of KD. Tumour specimens of our case showed vascular endothelial cell swelling and intratumour vessel thrombosis with leucocyte infiltration. These findings were consistent with vasculitis. In KD, inflammatory cytokines such as IL-1 and TNF-alpha play a major role in developing a vasculitis [1]. Unfortunately, determination of IL-1 or TNF-alpha level was not done. Nevertheless, vasculitis due to KD possibly caused the rapid swelling of the hidden tumour and the subsequent acute urinary retention. Paediatricians should look for this type of acute urinary retention in patients with KD.

TRAIL/Apo2L Ligands Induce Apoptosis in Malignant Rhabdoid Tumor Cell Lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a potent inducer of apoptosis in various cancer cells, whereas normal cells are not sensitive to TRAIL-mediated apoptosis. Four TRAIL/Apo2L receptors (DR4, DR5, DcR1, and DcR2) have been identified. DR4 and DR5 have a death domain, whereas DcR1 and DcR2 are called decoy receptors because of their incomplete or lack of a death domain. Malignant rhabdoid tumor (MRT) is an aggressive neoplasm showing a poor prognosis because of its resistance to chemotherapeutic agents. In this study, we examined whether TRAIL could induce apoptotic cell death in MRT cell lines. We found that although half of the MRT cell lines examined were sensitive to TRAIL/Apo2L, Western blot analysis revealed that the expression of DcR2 was low in TRAIL-sensitive MRT cells. We examined the effect of doxorubicin on the expression levels of TRAIL receptors and its enhancement on the susceptibility of MRT cell lines to TRAIL. Western blot and flow cytometric analyses revealed that doxorubicin significantly increased the expression of DR5, and somewhat up-regulated the expression of DR4 and DcR2. Moreover, doxorubicin, NF-κB inhibitor (SN50), and PI3-kinase/Akt inhibitor (wortmannin, LY294002) enhanced the susceptibility of MRT cell lines to TRAIL/Apo2L-induced apoptosis. These results suggest that TRAIL/Apo2L may provide the basis for clinical trials of TRAIL-based treatment to improve the outcome of MRT patients.

Malignant rhabdoid tumor shows a unique neural differentiation as distinct from neuroblastoma

Malignant rhabdoid tumors (MRT) show a multiphenotypic diversity, including a neural phenotype. To elucidate the difference in neural characteristics between MRT and neuroblastoma, we examined the expression of synapsin I, neuron‐restrictive silencer factor (NRSF), neurofilament medium‐size (NF‐M) and chromogranin A (CGA) in five MRT cell lines (TM87–16, STM91–01, TTC549, TTC642 and YAM‐RTK1) and five neuroblastoma cell lines under differentiation‐induction with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Our results showed TM87–16 and TTC642 cells, expressed synapsin I and NF‐M before TPA induction, had a neural phenotype. After differentiation‐induction, only TM87–16 cells expressed CGA. Among all neuroblastoma cells, expression of NF‐M and CGA was stable at a high level throughout TPA‐induced differentiation. In TM87–16 and TTC642 MRT cells, synapsin I mRNA promptly increased after TPA differentiation, with the peak level at 6 h, and thereafter, synapsin I mRNA rapidly decreased in a time‐dependent manner. The decreased expression of synapsin I correlated with an increased expression of NRSF during differentiation‐induction. In contrast, in some neuroblastoma cells, a significant up‐regulation of synapsin I was observed concurrently with a down‐regulation of NRSF. The inverse relationship between NRSF and synapsin I expression in TM87–16 and TTC642 MRT cells was opposite to that of neuroblastoma cells. Our results showed that the neural characteristics of these MRT cells are fairly distinct from those of neuroblastoma cells. These MRT cells appeared to have only limited capability for neural differentiation, and were still in an extremely early stage of neural differentiation. (Cancer Sci 2003; 94: 37–42)

Estrogen receptor expression and estrogen receptor-independent cytotoxic effects of tamoxifen on malignant rhabdoid tumor cells in vitro.

Recent studies have shown that the antiestrogen tamoxifen (TAM) can be used in the treatment of malignant neoplasms other than breast cancer. In the present study, we investigated the expression of estrogen receptor (ER) in six malignant rhabdoid tumor (MRT) cell lines. Alterations in MRT cell growth in response to estrogen or antiestrogens (4‐hydroxytamoxifen (4‐OHT), TAM, and ICI 182 780) were also investigated. RT‐PCR and western blotting showed that ER‐a was expressed in three of the six MRT cell lines. While 17‐β‐estradiol (E2) did not significantly alter MRT cell line proliferation, the hydroxylated tamoxifen metabolite 4‐OHT significantly inhibited the growth of all 6 MRT cell lines. However, the steroidal antiestrogen ICI 182 780 did not alter the proliferation of any of the MRT cell lines. 4‐OHT induced apoptosis in both ER‐α‐negative and ER‐α‐positive MRT cell lines, as assessed by nuclear morphology and DNA fragmentation. Neither growth inhibition nor induction of apoptosis due to 4‐OHT was blocked by the addition of excess E2. Our data suggested that 4‐OHT induced cytotoxic effects against MRT cells, and that these effects were independent of ER expression.

Expression of pericyte, mesangium and muscle markers in malignant rhabdoid tumor cell lines: Differentiation‐induction using 5‐azacytidine

Malignant rhabdoid tumor (MRT) has been considered to have multiphenotypic diversity characteristics. Some MRTs exhibit a neural phenotype. However, it is still unclear whether MRT cells can display a skeletal muscle, smooth muscle or smooth muscle‐like cell phenotype, like those of pericytes and mesangial cells. To determine if MRTs exhibit skeletal muscle cell or smooth muscle‐like cell phenotypes, six MRT cell lines (TM87‐16, STM91‐01, TTC549, TTC642, YAM‐RTK1 and TTC1240) were examined for markers of skeletal muscle (MyoD, myogenin, myf‐5, myf‐6, acetylcholine receptor‐α, ‐β and ‐γ), smooth muscle (α‐smooth muscle actin, SM‐1 and SM22), and smooth muscle‐like cells, such as pericytes (angiopoietin‐1 and ‐2) and mesangial cells (megsin), using conventional RT‐PCR, semi‐quantitative PCR, western blotting and immunocytochemistry before and after differentiation‐induction with 5‐azacytidine. α‐Smooth muscle actin and SM22 were detected in all six MRT cell lines, while MyoD and myf‐5, crucial markers for skeletal myogenic determination, were not. The TM87‐16 cell line expressed SM‐1 and angiopoietin‐1. TTC1240 also expressed angiopoietin‐1. Interestingly, STM91‐01 expressed megsin, a novel marker for mesangial cells, in addition to angiopoietin‐1. Our results indicated that some MRTs exhibited smooth muscle and/or smooth muscle‐like cell phenotypes and some renal MRTs might be of mesangial origin. Recently, smooth muscle and also smooth muscle‐like cells have been considered to be of neuroectodermal origin. MRT can thus considered to belong to the category of primitive neuroectodermal tumors (PNETs) in the broad sense.

Malignant rhabdoid tumor shows incomplete neural characteristics as revealed by expression of SNARE complex.

To elucidate the biological differences in neural phenotype between malignant rhabdoid tumor (MRT) and neuroblastoma cell lines, we examined the expression of solube N‐ethylmaleimide‐sensitive fusion protein attachment protein receptor (SNARE) complex proteins in MRT cell lines under differentiation induction with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Six MRT cell lines (TM87‐16, STM91‐01, TTC642, TTC549, YAM‐RTK1, and TTC1240) and six neuroblastoma cell lines (IMR‐32, NH12, SCCH26, TGW, NB‐1, and NB‐NR) were used in this study. Expression of SNAREs: the vesicle SNARE (synaptotagmin, synaptophysin, and synaptobrevin‐2) and the target SNARE (syntaxin 1A, SNAP‐25A/B) was examined. Our results showed that in MRT cells, only two cell lines (TM87‐16, TTC642) expressed the vesicle SNARE and the target SNARE with the exception of SNAP‐25B, while all neuroblastoma cells expressed the entire SNARE complex. During differentiation, synaptotagmin was upregulated in these two MRT cell lines. Interestingly, synaptophysin was downregulated in these MRT cell lines in contrast with the neuroblastoma cell lines. SNAP‐25B was not expressed in MRT cells after differentiation with TPA. MRT cells having a neural phenotype morphologically looked like neuroblastoma cells after treatment with TPA. However, the expression of SNARE complex was incomplete in MRT cells. Our results suggest that the biological characteristics of MRT cells with neural phenotype are distinct from those of neuroblastoma cells.

Increased apoptosis and hypomyelination in cerebral white matter of macular mutant mouse brain

Hypomyelination in developing brain is often accompanied by congenital metabolic disorders. Menkes kinky hair disease is an X-linked neurodegenerative disease of impaired copper transport, resulting from a mutation of the Menkes disease gene, a transmembrane copper-transporting p-type ATPase gene (ATP7A). In a macular mutant mouse model, the murine ortholog of Menkes gene (mottled gene) is mutated, and widespread neurodegeneration and subsequent death are observed. Although some biochemical analysis of myelin protein in macular mouse has been reported, detailed histological study of myelination in this mouse model is currently lacking. Since myelin abnormality is one of the neuropathologic findings of human Menkes disease, in this study early myelination in macular mouse brain was evaluated by immunohistochemistry. Two-week-old macular mice and normal littermates were perfused with 4% paraformaldehyde. Immunohistochemical staining of paraffin embedded and vibratome sections was performed using antibodies against either CNPase, cleaved caspase-3 or O4 (marker of immature oligodendrocytes). This staining showed that cerebral myelination in macular mouse was generally hypoplastic and that hypomyelination was remarkable in internal capsule, corpus callosum, and cingulate cortex. In addition, an increased number of cleaved caspase-3 positive cells were observed in corpus callosum and internal capsule. Copper deficiency induced by low copper diet has been reported to induce oligodendrocyte dysfunction and leads to hypomyelination in this mouse model. Taken together, hypomyelination observed in this study in a mouse model of Menkes disease is assumed to be induced by increased apoptosis of immature oligodendrocytes in developing cerebrum, through deficient intracellular copper metabolism.