Loss of density-dependent growth inhibition and dissociation of alpha-catenin from E-cadherin.

Normal human breast epithelial (HBE) cells at early (9th) passage ceased growth and formed a monolayer when they reached confluence. Immunostaining and Western blotting revealed that alpha- and beta-catenins colocalized and coprecipitated with E-cadherin, suggesting a complex formation of E-cadherin with alpha- and beta-catenins in early passage cells. In contrast, HBE cells at late (12-13th) passage did not cease growth after confluence but stratified. The late passage cells exhibited enhanced colony forming ability in soft agar compared with early passage cells, however, they had a definite proliferating lifespan and were primarily diploid. In late passage cells grown as multilayers, alpha-catenin was expressed but did not colocalize or coprecipitate with E-cadherin, suggesting its dissociation from E-cadherin. Coimmunoprecipitation of alpha-actinin with alpha-catenin suggested an indirect link between the E-cadherin-beta-catenin complex and alpha-actinin via alpha-catenin in early, but not in late passage cells. Beta-Catenin in late passage cells was tyrosine phosphorylated and was not dephosphorylated following the addition of inhibitors of tyrosine kinases. Inhibition of dephosphorylation of beta-catenin in early passage cells by vanadate, an inhibitor of protein tyrosine phosphatases, caused overgrowth of cells beyond the saturation density and loss of alpha-catenin from the E-cadherin-beta-catenin complex. The results suggest that E-cadherin requires its association with alpha-actinin-associated alpha-catenin to maintain epithelial monolayers and accomplish the density-dependent inhibition of growth. In addition, association between E-cadherin and alpha-catenin is suggested to be prevented by the presence of tyrosine phosphorylated beta-catenin which associates with E-cadherin.

Induction of tyrosine phosphorylation and association of beta-catenin with EGF receptor upon tryptic digestion of quiescent cells at confluence.

Normal human breast epithelial (HBE) cells which reached confluence ceased growth and tightly adhered to each other, forming a monolayer. In quiescent cells thus arrested by density, E-cadherin colocalized and coimmunoprecipitated with alpha- and beta-catenins in the boundary region between adjacent cells. By contrast, immunocytostaining and Western blot analyses revealed that E-cadherin colocalized and coprecipitated with beta-catenin but not with alpha-catenin in exponentially growing cells at low density. As a comparable amount of alpha-catenin was detected in the total cell lysate of cells at different densities, it is suggested that alpha-catenin is present but dissociates from the E-cadherin-beta-catenin complex in growing cells. beta-Catenin was tyrosine phosphorylated in growing cells at low density but not in quiescent cells at confluence. Tyrosine phosphorylation of beta-catenin was concomitantly induced with association of beta-catenin with EGF receptor (EGFR) when quiescent cells at confluence were dissociated into single cells by tryptic digestion, being accompanied by dissociation of alpha-catenin from E-cadherin. Both tyrosine phosphorylation and association of beta-catenin with EGFR were inhibited by tyrphostin, a specific inhibitor of the EGFR tyrosine kinase, whereas dissociation of alpha-catenin from E-cadherin was not. The results suggest that tyrosine phosphorylation of beta-catenin is achieved by EGFR upon tryptic digestion of cells and concurrent with but independent of dissociation of alpha-catenin from E-cadherin. beta-Catenin thus phosphorylated at tyrosine is suggested to play the role in preventing alpha-catenin once dissociated from reassociating with E-cadherin until cells reach confluence.

Relationship between radical production and natural killer cytotoxic factor (NKCF) in canine natural killer (NK) cell-mediated cytotoxicity.

The relationship between radical production and natural killer cytotoxic factor (NKCF) release via canine natural killer (NK)-mediated cytotoxic mechanism was examined. Radical production and NKCF release was induced in NK cells stimulated with either dead target cells, or their cytoplasmic membranes, as well as live target cells. Canine NKCF evoked target cell lysis but did not induce radical production. Radical production was inhibited by the addition of Tiron or n-propyl gallate, whereas NK-mediated cytotoxicity and NKCF release were only inhibited by the addition of n-propyl gallate. These results suggested that radical production and NKCF release may be induced by the contact and binding of NK cells to the target cell cytoplasmic membrane. Therefore, the release of NKCF from NK cells attached to the target cell cytoplasmic membrane may be associated with the production of radicals, especially hydroxyl radicals.

[Diagnostic accuracy of 201T1 SPECT.MRI in brain diseases and inter-reader variance of diagnostic performance].

Fifteen-four studies of 201T1 brain tumor SPECT were independently interpreted by 9 nuclear medicine physicians with and without reference magnetic resonance images in 2 separate sessions to define an effect of referring images, and inter-observer variations. The physicians were requested to detect foci of abnormal deposits, and to discriminate whether they were malignant or not according to 5-grade scaling of subjective diagnostic confidence. Receiver-operating characteristics (ROC) analysis was performed. Mean sensitivity for presence of lesions (SEP), and sensitivity and specificity for malignancy of 201T1 SPECT were 84, and 53 and 55%, which were changed to 94, and 74 and 55% after referring to the MR images. The SFP was significantly improved (p < 0.05), but sensitivity and specificity for malignancy, assessed by areas under the ROC curves, were not significantly improved. Inter-observer variation of the SFP was significantly reduced with addition of the referring MR images. Cerebral lobar localization of lesions by SPECT showed great inter-observer variations (true localization ranged from 30 to 68.4%). It is concluded that 201T1 brain tumor SPECT has moderate sensitivity and specificity for malignancy, which is not improved by addition of anatomical reference images, that additional MR images reduce inter-observer variation of confidence on lesion presence, and that SPECT localization of lesions has great inter-observer variations.

Inhibition of demecolcin-induced DNA synthesis by inhibitors of phospholipase C and protein kinase C.

Exposing normal human breast epithelia (HBE) cells, which were growth arrested by a 3-day culture in EGF-deprived medium, to the microtubule disrupting agent, demecolcin (N-deacetyl-N-methyl-colchicine), for 2 hr significantly stimulated the initiation of DNA synthesis 22 hr later. The demecolcin-induced DNA synthesis was not accompanied by activation of the EGF receptor and it was inhibited by calphostin C, an inhibitor of protein kinase C (PKC), and U-73122, an inhibitor of phospholipase C (PLC). Contrary to this, the EGF-induced DNA synthesis was inhibited by tyrphostin A25, a specific inhibitor of the EGF receptor tyrosine kinase, and calphostin C. The results suggested that the involvement of PLC and PKC in the demecolcin-induced signal transduction pathway leads to the initiation of DNA synthesis.

Characterization of natural killer cytotoxic factor (NKCF) from canine NK cells.

We investigated the presence of canine natural killer cytotoxic factor (NKCF). Canine natural killer (NK) cell-mediated cytotoxicity measured by 51chromium (51Cr) release assay was found to be highest in the T-cell population, which was fractionated into the 35-40% Percoll fraction by discontinuous gradient centrifugation. The cytotoxicity of NKCF in the culture supernatant showed a similar tendency to NK activity. Release of NKCF was rapid after contact with target cells, and reached a plateau in 60 min. The cytotoxicity of NKCF could be detected within at least 15 min in coculture with CL-1 target cells, reaching a plateau in 60 min. We also characterized canine NKCF and found it to be a protein, which was stable against both heat and cold treatment. These findings suggest that canine NK cells release NKCF immediately after recognition and binding to the target cell, and that NKCF plays an important role in canine NK-mediated cytotoxicity.

Release of natural killer cytotoxic factor (NKCF) from canine natural killer (NK) cells stimulated with cytoplasmic membrane of target cells.

The presence of canine natural killer cytotoxic factor (NKCF) and the mechanism of the release of the NKCF by canine natural killer (NK) cells were studied. The cytotoxicity in the culture supernatant of effector cells cultured with target cells was dependent on the count of NK cells. Therefore, this suggests that the cytotoxic factor in this culture supernatant is NKCF. The NK-sensitive live target cells stimulated the release of NKCF from NK cells, but the NK-insensitive target cells did not. Moreover, NKCF was also released from NK cells stimulated with either killed target cells or their cytoplasmic membrane as well as live target cells. These findings suggest that the structure of the cytoplasmic membrane of target cells is involved in the recognition, binding and the release of NKCF by NK cells.

[Exercise and rest myocardial scintigraphy with 201TlCl/99mTc-MIBI dual energy acquisition using triple-energy window scatter correction].

We carried out dual 201Tl/99mTc-MIBI imaging, to reduce the time required for exercise myocardial scintigraphy. We investigated 4 different protocols. In protocol (A), Tl was injected at rest followed by the injection of MIBI at peak exercise. Dual SPECT images were obtained by 201Tl/99mTc simultaneous acquisition. Protocol (B) means reverse either, in which MIBI was injected at rest followed by the administration of Tl at peak exercise. In protocol (C), exercise was performed first with MIBI-injection, and then Tl was injected at rest after one hour later. Simultaneous acquisition was also performed. In protocol (D), after the rest Tl-imaging, MIBI was injected at peak exercise, and then the MIBI-imaging was done. In protocol (A), (B) and (C), simultaneous acquisition was performed using TEW (Triple-Energy Window) scatter correction. Thanks to using dual isotopes, all procedures could be completed within 1-2 hours, which was much shorter than the conventional myocardial perfusion imaging. Scatter correction was useful for accurate diagnoses, when the simultaneous imaging is performed.

Clinical significance of antibodies to native or denatured 60-kd or 52-kd Ro/SS-A proteins in Sjögren's syndrome.

OBJECTIVE: To evaluate the clinical significance of antibodies to native or denatured (anti-n or anti-d) 60- or 52-kd Ro/SS-A proteins (60K or 52K) in Sjögren's syndrome (SS). METHODS: The presence of antibodies to denatured and native Ro/SS-A proteins was determined by immunoblotting and immunoprecipitation, respectively. Salivary gland dysfunction was evaluated by salivary function scintigraphy. RESULTS: The incidence of anti-d-60K without anti-d-52K was lower among patients with systemic lupus erythematosus with SS (SLE/SS) and among those with primary SS, compared with patients who had SLE without SS, whereas anti-d-52K without anti-d-60K was more common in SLE/SS patients and primary SS patients than in SLE patients without SS. All of the patients with anti-Ro/SS-A had anti-n-60K. Serologic abnormalities and salivary gland dysfunction were associated with anti-n-60K in SS, whereas Hashimoto's thyroiditis in SS was related to anti-d-60K. Anti-d-52K was not associated with any extraglandular or glandular symptoms in SS. CONCLUSION: The data indicate that anti-n-60K, which appears to recognize conformational epitopes, is associated with clinical features of SS characterized by glandular dysfunction.

Relationship between autoantibodies and clinical parameters in Sjögren's syndrome.

Glandular function as estimated by salivary function scintigraphy and extraglandular manifestations were compared among 174 Sjögren's syndrome (SS) patients according to their anti-Ro/SSA, anti-La/SSB, and anti-U1RNP autoantibody status, to clarify the relationship between these autoantibodies and clinical parameters in SS. These antibodies were detected by RNA-immunoprecipitation. Anti-La/SSB or only anti-Ro/SSA antibody was common in 84 primary SS (P-SS) patients, whereas the frequency of only anti-U1RNP was high in 90 secondary SS (S-SS) patients, especially in those with systemic lupus erythematosus (SLE). Antibody-negativity was common in SS with rheumatoid arthritis and was also found in 33% of P-SS. In P-SS, salivary gland dysfunction and parotid swelling were severe in patients who had serological abnormalities with anti-Ro/SSA and with or without anti-La/SSB. They were mild in antibody-negative patients who had mild extraglandular symptoms and in patients with only anti-U1RNP antibody who had Raynaud's phenomenon, pulmonary fibrosis, and later disease onset. P-SS patients positive for both anti-Ro/SSA and anti-U1RNP had SLE-like features. SS could be classified clinically according to these autoantibodies.

[Phase I clinical study of 99mTc-ECD].

A phase I clinical study of 99mTc-L,L-ethyl cysteinate dimer (99mTc-ECD) was carried out in 3 normal volunteers. There was no significant change in vital signs and laboratory parameters attributing to the radiopharmaceutical. 99mTc-ECD was rapidly taken up by the brain, reaching the maximum peak activity within 1 min after the injection and remained relatively constant over several hours. The brain uptake of 99mTc-ECD at 5 min was 5.4 +/- 0.5% which decreased to 5.0 +/- 0.3% by 65 min. 99mTc-ECD cleared rapidly from other organs. The primary excretion route was through the kidneys. Cumulative 99mTc activity in the urine at 90 min and 24 hr were 60.2 +/- 7.3% and 88.5 +/- 10.3%, respectively. The critical organ was the bladder wall with an estimated radiation dose of 0.073 mGy/MBq, which was acceptable value. Clear SPECT images were obtained at 30, 90 and 150 min postinjection. In conclusion, 99mTc-ECD is a safe and promising radiopharmaceutical for the evaluation of regional cerebral blood flow.

[Immunoscintigraphy of colorectal cancer with 111In labeled anti-CEA monoclonal antibody (ZCE-025)].

Clinical trials with 111In labeled anti-CEA monoclonal antibody (ZCE-025) was initiated. Five patients with colorectal cancer suspected were given an intravenous injection of 1 mg of 111In labeled ZCE-025. Planar and SPECT images were obtained 24 and 72 hours after injection. Surgical operation was performed on all patients between 7 and 10 days post injection. Of 4 primary sites, all were clearly visualized. Intrahepatic metastasis was visualized as higher activity than normal liver in one of two patients. In one patient whose imaging was negative, no residual cancer was found at surgery. Persistent accumulation of 111In in the lymph nodes was also observed in one patient. Surgical exploration of these lymph nodes showed no gross or microscopic evidence of metastases of colon cancer. No side effects were encountered, although HAMA were detected in all 5 patients by 4 weeks after the administration of ZCE-025. Immunoscintigraphy appears useful in distinguishing recurrent tumor from postoperative granuloma. Further investigation directed to the causes of 111In accumulation in tumor-free lymph nodes is required.

Usefulness of pinhole collimator in differential diagnosis of metastatic disease and degenerative joint disease in the vertebrae; evaluation by receiver operating characteristics (ROC) analysis.

In order to evaluate the diagnostic efficacy of pinhole collimator (PHC) imaging combined with an X-ray for vertebral metastasis, our prospective study has employed receiver operating characteristics (ROC) analysis in 21 patients, 11 with osseous metastasis and 15 with degenerative joint disease in the lumbar vertebrae. PHC imaging provided better anatomic information on the extent of 99mTc-MDP accumulation. PHC vertebral scintigraphy had a considerable impact on the decision-making process, although with variations and not very satisfactory results among the physicians with little experience. Our study suggests that PHC imaging and X-ray film are useful in differentiating between osseous metastasis and degenerative joint disease in the vertebra.