A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission.

The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo.

Relationship between time after intake of grapefruit juice and the effect on pharmacokinetics and pharmacodynamics of nisoldipine in healthy subjects.

A clinical study was performed in eight healthy volunteers to investigate the effect of various timing of grapefruit juice intake on nisoldipine pharmacokinetics and pharmacodynamics, and to validate our pharmacokinetic model. The subjects were given 10 mg oral nisoldipine with water (control), or 5 mg oral nisoldipine with 200 mL grapefruit juice (G0) or with water at 14 (G14), 38 (G38), 72 (G72) or 96 hours (G96) after a 7-day period of thrice-daily intake of grapefruit juice. Grapefruit juice ingestion did not affect heart rate or the effect area during the first 8 hours of heart rate after nisoldipine administration, although significant decreases of systolic and diastolic blood pressure were caused in G0 by coadministration of grapefruit juice with nisoldipine. Headaches were reported by 3, 2, and 1 persons in G0, G14, and G38, respectively, but no subjects in G72 and G96 reported headaches. Compared with the control group, the maximum plasma concentration of nisoldipine was significantly increased after grapefruit juice intake in G0 and G14, and the plasma concentration was significantly increased at each time in G0 to G72. Therefore the effect of grapefruit juice decreased time dependently and lasted for at least 3 days after intake. Furthermore, our model gave predicted values in good agreement with the observed values. It is therefore necessary to withhold grapefruit juice for at least 3 days before administration of the drug to prevent grapefruit juice-nisoldipine interaction.

Frequent co-expression of the HOXA9 and MEIS1 homeobox genes in human myeloid leukemias.

There is increasing evidence that HOX homeobox genes play a role in leukemogenesis. Recent studies have demonstrated that enforced co-expression of HOXA9 and MEIS1 in murine marrow leads to rapid development of myeloid leukemia, and that these proteins exhibit cooperative DNA binding. However, it is unclear whether co-activation of HOXA9 and MEIS genes is a common occurrence in human leukemias. We surveyed expression of HOXA9 and MEIS1 in 24 leukemic cell lines and 80 patient samples, using RNase protection analyses and immunohistochemistry. We demonstrate that the expression of HOXA9 and MEIS1 in leukemia cells is uniquely myeloid, and that these genes are commonly co-expressed in myeloid cell lines and in samples of acute myelogenous leukemia (AML) of all subtypes except in promyelocytic leukemia. While HOXA9 is expressed in most cases of chronic myelogenous leukemia, MEIS1 is weakly expressed or not at all. Immunohistochemical staining of selected AML samples showed moderate to high levels of HOXA9 protein, primarily cytoplasmic, in leukemic myeloblasts, with weaker and primarily nuclear staining for MEIS1. These data support the concept that co-activation of HOXA9 and MEIS1 is a common event in AML, and may represent a common pathway of many different oncogenic mutations.

Retinoic acid increases tyrosine phosphorylation of focal adhesion kinase and paxillin in MCF-7 human breast cancer cells.

Treatment of estrogen receptor (ER)-positive MCF-7 human breast cancer cells with retinoic acid (RA) inhibited cell growth and increased cell adhesion to fibronectin. In contrast, ER- MDA-MB-231 cells failed to respond. Western blot analysis showed that tyrosine phosphorylation of two major bands at Mr 125,000 and Mr 68,000 was induced by RA in ER+ MCF-7 human breast carcinoma cells. However, this induction was a late phenomenon detectable at 12 and 24 h, but not within 3 h. A similar increase of tyrosine phosphorylation by RA was observed in ER+ human breast cancer cell lines T-47D and ZR-75-1, but not in the ER- cell lines MDA-MB-231, MDA-MB-453, and MDA-MB-468. Focal adhesion kinase and paxillin, which localize in focal adhesion plaques and may play important roles in the integrin signaling pathway, were identified as the major proteins showing RA-induced tyrosine phosphorylation. The retinoid X receptor-selective compound SR11237 failed to induce tyrosine phosphorylation, indicating that retinoid X receptor activation is not involved in this phenomenon. In contrast, stable overexpression of a truncated RA receptor (RAR) alpha cDNA, RARalpha403, with strong RAR dominant negative activity prevented the increase in tyrosine phosphate, suggesting that RAR signaling is involved in RA-induced tyrosine phosphorylation. Tyrosine phosphorylation was induced the most by the RAR-alpha (193836), followed by RAR-gamma (194433), but was not significantly induced by RAR-gamma (193174)-selective retinoids. This study demonstrates a coordinated albeit relatively late effect of RA on cell adhesion and tyrosine phosphorylation in ER+ human breast cancer cells and suggests RAR-alpha as the major responsible retinoid receptor.

EGF-Receptor phosphorylation and signaling are targeted by H2O2 redox stress.

Inflammation of the respiratory tract is associated with the production of reactive oxygen species, such as hydrogen peroxide (H2O2) and superoxide (O2-), which contribute extensively to lung injury in diseases of the respiratory tract. The mechanisms and target molecules of these oxidants are mainly unknown but may involve modifications of growth-factor receptors. We have shown that H2O2 induces epidermal growth factor (EGF)-receptor tyrosine phosphorylation in intact cells as well as in membranes of A549 lung epithelial cells. On the whole, total phosphorylation of the EGF receptor induced by H2O2 was lower than that induced by the ligand EGF. Phosphorylation was confined to tyrosine residues and was inhibited by addition of genistein, indicating that it was due to the activation of protein tyrosine kinase (PTK). Phosphoamino acid analysis revealed that although the ligand, EGF, enhanced the phosphorylation of serine, threonine, and tyrosine residues, H2O2 preferentially enhanced tyrosine phosphorylation of the EGF receptor. Serine and threonine phosphorylation did not occur, and the turnover rate of the EGF receptor was slower after H2O2 exposure. Selective H2O2-mediated phosphorylation of tyrosine residues on the EGF receptor was sufficient to activate phosphorylation of an SH2-group-bearing substrate, phospholipase C-gamma (PLC-gamma), but did not increase mitogen-activated protein (MAP) kinase activity. Moreover, H2O2 exposure decreased protein kinase C (PKC)-alpha activity by causing translocation of PKC-alpha from the membrane to the cytoplasm. These studies provide novel insights into the capacity of a reactive oxidant, such as H2O2, to modulate EGF-receptor function and its downstream signaling. The H2O2-induced increase in tyrosine phosphorylation of the EGF receptor, and the receptor's slower rate of turnover and altered downstream phosphorylation signals may represent a mechanism by which EGF-receptor signaling can be modulated during inflammatory processes, thereby affecting cell proliferation and thus having implications in wound repair or tumor formation.

H2O2 acts on cellular membranes to generate ceramide signaling and initiate apoptosis in tracheobronchial epithelial cells.

Hydrogen peroxide (H2O2) is an inflammatory oxidant which contributes to the pathogenesis of chronic diseases such as lung injury of the respiratory tract, atherosclerosis and cancer. The mechanisms and target sites of this reactive oxidant are mainly unknown. So far there are opposing reports as to whether reactive oxidants inhibit or promote apoptosis. We activated the death pathway in primary tracheobronchial epithelial (TBE) cells with H2O2 (20-200 microM) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation associated with apoptosis. Elevation of ceramide with exogenous ceramide analogs was sufficient for apoptosis induction with the same characteristics and in the same time frame. H2O2 induced rapid sphingomyelin hydrolysis to ceramide, the elevation of which paralleled the induction of apoptosis. Furthermore, H2O2 acted directly on TBE cells membrane preparations devoid of nuclei, stimulating sphingomyelin hydrolysis through a neutral Mg2+ dependent sphingomyelinase (SMase). These data suggest that the formation of ceramide from sphingomyelin in the plasma membrane is a key event in H2O2-induced apoptosis in tracheobronchial epithelial cells.

EGF receptor phosphorylation is affected by ionizing radiation.

Eukaryotic cells respond to ionizing radiation with cell cycle arrest, activation of DNA repair mechanisms, and lethality. However, little is known about the molecular mechanisms that constitute these responses. Here we report that ionizing radiation enhances epidermal growth factor (EGF) receptor tyrosine phosphorylation in intact cells as well as in isolated membranes of A431 cells. Phosphoamino acid analysis revealed that ionizing radiation preferentially enhances tyrosine phosphorylation, while EGF enhances the phosphorylation of all three phosphoamino acids (serine, threonine and tyrosine) of the EGF receptor. In addition, radiation reduces the turnover rate of the EGF receptor, while EGF increases the rate of the receptor turnover and down-regulation. Moreover, the confined radiation-induced phosphorylation of tyrosine residues is inhibited by genistein, indicating that this phosphorylation of EGF receptor is due to protein tyrosine kinase activation. These studies provide novel insights into the capacity of radiation to modulate EGF receptor phosphorylation and function. The radiation-induced elevation in the EGF receptor tyrosine phosphorylation and the receptor's slower rate of turnover are discussed in terms of their possible role in cell growth and apoptosis modulation.

Retinoic acid inhibition of cell cycle progression in MCF-7 human breast cancer cells.

Cell cycle analysis indicates that retinoic acid (RA) inhibition of MCF-7 cell growth occurs through induction of G1 arrest with a concomitant reduction in the proportion of cells in S and G2 + M phases. RA did not affect cyclins D1, A, and E and cyclin-dependent kinase 2 (CDK2) expression, but significantly reduced cyclin D3 and CDK4 expression after 24 h. RA also inhibited cyclin B1 and CDC2 expression, possibly responsible for the reduction of the proportion of cells in G2 + M and S phases. RA did not induce p16 and p27 expression, but obviously reduced p21 level in MCF-7 cells. The retinoid markedly reduced pRB protein level and abrogated pRB phosphorylation after 48 h; it also reduced transcription factor E2F1 expression at both the mRNA and protein levels. E2F1 promoter activity was reduced by 60%, which is probably responsible, at least in part, for the reduction of E2F1 expression in RA-treated MCF-7 cells. These observations demonstrate a marked effect of RA on some of the key cell cycle regulatory proteins in MCF-7 cells. Cyclin D3 and CDK4 are likely the early targets of RA, followed by reduced pRB expression and phosphorylation, as well as by the inhibition of the E2F1 transcription factor which controls progression from G1 to S phase. Most of these events precede the observed reduction in MCF-7 cell growth, which begins at Day 3 of RA treatment.

Induction of heat shock 70 mRNA by cadmium is mediated by glutathione suppressive and non-suppressive triggers.

The induction mechanism of heat shock 70 (Hsp70) gene by cadmium was investigated. In human amniotic WISH cells, Hsp 70 was induced by cadmium in a dose- and time-dependent manner. Cadmium-induced Hsp70 mRNA levels were enhanced 3- to 4-fold after depletion of intracellular glutathione (GSH) by either diethylmaleate or buthionine sulfoximine. Under these conditions, hydrogen peroxide might increase in the absence of substrate for glutathione peroxidase. We found that exogenous hydrogen peroxide alone induced Hsp70 which was further enhanced significantly after GSH-depletion by diethylmaleate. On the other hand, treatment of cells by diethyldithiocarbamate, an inhibitor of superoxide dismutase, induced Hsp70 2-fold over the level of control. This induction was further stimulated by cadmium even in the presence of GSH. Furthermore, a 4-fold increase of intracellular GSH by the treatment of cells with glutathione isopropyl ester did not diminish the cadmium-induced Hsp70. Gel mobility shift assays of nuclear extracts, from these differently treated cells, with oligonucleotide containing a promoter region of Hsp70 gene revealed that the levels of Hsp70 mRNA observed in the present study corresponded to the changes of transcription. These results imply that the induction of Hsp70 mRNA by cadmium is mediated at least partly via reactive oxygen species and attenuated by cellular GSH and that some part of cadmium-induced Hsp70 can not be eliminated by GSH, suggesting that multiple signals are functioning for this induction.

Microscopic ovarian metastasis of the uterine cervical cancer.

Six hundred forty-seven cases of carcinoma of the uterine cervix with FIGO stages Ib or more were initially treated with hysterectomy at Kyushu University Hospital from 1973 to 1987. In these, 597 cases could be pathologically reviewed for ovarian metastasis. In these 597 cases, 335 were stage Ib, 71 IIa, 185 IIb, and 6 IIIb. Only 3 (0.5%) of 597 showed ovarian metastasis. All 3 cases were stage IIb. None of stage Ib cancer cases had ovarian metastasis. One (0.19%) of 524 squamous cell carcinomas metastasized to the ovary, whereas 2 (5.5%) of 36 pure adenocarcinomas revealed ovarian metastasis. Interestingly, all ovarian metastatic lesions were microscopic in size and found in the ovarian hilus. As for the primary lesion, all cases with ovarian metastasis showed deep myometrial invasion, corpus invasion, and lymphatic permeation. Two cases showed pelvic lymph node metastases and positive peritoneal washing cytology. From the results of our study, it can be said that it is fairly safe to preserve the ovary at the time of radical operation in squamous cell carcinoma of the uterine cervix, but it may not be safe to preserve the ovary in pure adenocarcinoma of the uterine cervix.

[Mesenteric hematoma; a pediatric case].

A 3-Year-old girl was admitted to National Beppu Hospital due to abdominal mass. US examination revealed cystic mass with multiple scattered nonreverberatory echoes in right upper abdomen. CT examination revealed almost homogeneous mass (29-31 H.U.) with a few internal components (36-42 H.U.). US and CT were useful methods for diagnosis of mesenteric hematoma.

[A serous cystadenoma of the ovary of borderline malignancy with a fifteen-year history. A case report].

A case of a serous cystadenoma of a ovary of borderline malignancy is reported. Sixteen years earlier, the patient had undergone an exploratory laparotomy because of ovarian tumor, and the histologic diagnosis had been a serous cystadenocarcinoma. Postoperative chemotherapy was not effective and drainage of the tumor fluid had been performed for 15 years, with the estimated drainage volume estimated to have reached, 1,000 1. Gradual malnutrition and marked tumor growth then become apparent. A reevaluation of the initial histologic slides and her clinical course strongly suggested a serous cystadenoma of borderline malignancy. Thus a tumor resection, a bilateral salpingo-oophorectomy, and a hysterectomy was performed. A histologic diagnosis of a resected specimen confirmed a serous cystadenoma of borderline malignancy and the histologic features were quite similar to those of the initial biopsy specimens. The patient is living well postoperatively for 8 months without postoperative chemotherapy.

Glassy cell carcinoma of the endometrium.

A case of glassy cell carcinoma of the endometrium in a 62-year-old woman is reported. Microscopically, a cytoplasm of ground-glass appearance was observed with a distinct cell wall and large nuclei containing prominent nucleoli. These histologic characteristics are consistent with those of glassy cell carcinoma of the cervix. Treatment consisted of total abdominal hysterectomy and bilateral salpingo-oophorectomy with pelvic lymph node dissection followed by external irradiation to the whole pelvis. The patient was alive without evidence of disease at 5 1/2 years.

[Surgical management of pulmonary metastasis from carcinoma of the uterine cervix].

The present study was designed to evaluate the efficacy of surgical management of pulmonary metastasis from carcinoma of the uterine cervix. We saw 609 cases of carcinoma of the uterine cervix from 1979 to 1987, and during the same period also saw 110 cases of recurrent carcinoma of the uterine cervix. Fourteen of these 110 cases were identified as having pulmonary metastasis, and in 11 of 14 cases the recurrent tumors were limited to the lung. Seven of these 11 cases underwent pulmonary resection. Six of the 7 survived more than 2 years after pulmonary resection. Among them, 1 patient has survived more than 4 years, and 1 patient more than 8 years. The tumor cells were thought to metastasize to the lung through the vertebral venous plexus (Batson's plexus) which was suggested as a metastatic route by Thomford et al. in their report on recurrent colon cancer. As a result of this study, if the recurrent tumor is clinically limited to the lung in patients with recurrent carcinoma of the uterine cervix, they should be treated by surgical resection of the pulmonary tumor.

Early adenocarcinoma of the uterine cervix--its histologic and immunohistologic study.

Eight cases of early adenocarcinoma selected from 101 adenocarcinomas of the uterine cervix were studied to establish the criteria of early adenocarcinoma. Lesions of these 8 cases were small in size. In 7 of 8 cases, these tumors originated in the area of the squamocolumnar junction (SCJ). Tumor cells consisted of two types of atypical columnar cells, i.e., tall columnar cells with enlarged and deeply eosinophilic cytoplasm and clear cells with enlarged and clear vacuoles. Tall columnar cells showed weak or negative reaction to high iron diamine (HID) stain and negative to Alcian blue (AB) stain. Clear cells showed negative reaction to HID stain and positive to AB stain. Although normal endocervical columnar cells showed markedly positive reaction to HID stain and negative reaction to AB stain, invasive adenocarcinoma cells showed similar reaction to early adenocarcinoma cells. From this study, it is surmised that early adenocarcinoma of the uterine cervix originates in the area of the SCJ and consists of tall cells in all cases and clear cells in 4 of 8 cases, and that HID-AB stain is useful in differentiating early adenocarcinoma cells from normal endocervical columnar cells.

Hyperreactio luteinalis in normal singleton pregnancy.

A case of hyperreactio luteinalis in a patient with normal singleton pregnancy is reported. The course of pregnancy had been normal until the 24th week of gestation, when the mother developed lower abdominal pain and signs of virilization. She delivered of a normal female infant at 39 weeks' gestation. The baby did not show any signs of masculinization. Serum testosterone, delta 4-androstene-dione, and 5 alpha-dihydrotestosterone of the mother were markedly elevated. They remained high after the delivery but returned to the normal ranges soon after the partial resection of the enlarged ovaries. Reported causes of hyperreactio luteinalis are reviewed. Their maternal serum androgen levels were compared with cases of luteoma of pregnancy.

The problem of stage Ia (FIGO, 1985) carcinoma of the uterine cervix.

The FIGO definition of stage Ia (microinvasive) carcinoma of the uterine cervix had been vague and caused continued confusion. In 1985 FIGO revised the definition of stage Ia by including measurements in the definition and it also subdivided stage Ia into stages Ia1 and Ia2. One hundred and eighteen of eight hundred and sixty patients with stage 0 to IIb carcinoma, who retrospectively satisfied the new FIGO definition of stage Ia, were reviewed with respect to depth of invasion, horizontal spread, number of invasive foci, vascular space involvement, pattern of invasion, incidence of lymph node metastasis, and clinical outcome. There were three potentially high-risk patients: one with pelvic lymph node metastases, one with parametrial metastasis, and the third with recurrence 23 months after initial treatment. It was pointed out that the border between stages Ia1 and Ia2 is very vague in the 1985 FIGO definition. It was proposed that a lesion with 3-mm or less stromal invasion and without vascular space involvement and confluency be defined as stage Ia1 and be safely treated with simple means.

Clinicopathologic study of squamous cell carcinoma of the ovary.

Clinicopathologic study was performed on 10 squamous cell carcinomas of the ovary. All four patients with stage II or III lesions had deteriorated within 1 year after the operation, and four of six patients with stage I lesions had survived over 5 years. Clinical findings of the patients with ovarian SCC, including age, chief complaint, clinical stage, and outcome of the patients, were similar to those of common epithelial cancer. The effectiveness of chemotherapy was not shown in this study. Histopathologic study revealed that squamous cell carcinoma may arise not only from epidermis, but also from squamous metaplastic epithelium of respiratory gland.

Carcinoma of the uterine cervix with variegated histological patterns and calcitonin production.

This is a report of a calcitonin-producing cervical cancer with variegated histological patterns. The levels of calcitonin correlated with the clinical course. Multiple lung metastases were completely controlled by cisplatin/Adriamycin/cyclophasphamide combination chemotherapy, although only for a short period. Microscopically, the tumor showed dominant small cell carcinoma; however, it did show differentiation into large cell nonkeratinizing carcinoma, keratinizing carcinoma, adenocarcinoma, and possibly carcinoid. Ultrastructural study revealed a few dense core secretory granules. Multiple immunohistochemical studies were performed. It is presumed that the tumor originated from the totipotent, immature precursor cells in the cervix and differentiated into endocrine carcinoma, squamous cell cancer, and adenocarcinoma.

Preoperative CT study of lymph nodes in cervical cancer--its correlation with histological findings.

A study was conducted to retrospectively evaluate the accuracy of abdominopelvic computed tomography (CT) in the diagnosis of paraaortic and pelvic lymph node metastases from carcinoma of the uterine cervix. Seventy patients with a diagnosis of invasive carcinoma of the cervix had preoperative CT of abdomen and pelvis and subsequently underwent a radical hysterectomy with pelvic lymph node dissection and paraaortic lymph node biopsy or an exploratory laparotomy with paraaortic lymph node biopsy. Five of six patients with metastatic paraaortic lymph nodes larger than 15 mm in diameter on the histologic slides were diagnosed by CT scan to have enlarged nodes. CT diagnosis was true-positive in five of seven patients with paraaortic lymph node metastases (71.4%). Two patients with false-positive paraaortic lymph nodes had clusters of small lymph nodes less than 10 mm in diameter on the histologic slides. In contrast, only a small number of the metastatic pelvic nodes were diagnosed by CT as enlarged nodes. CT diagnosis was true-positive in 5 of 11 sites with pelvic lymph node metastases (45.5%).