Multidrug resistance and the lung resistance-related protein in human colon carcinoma SW-620 cells.

BACKGROUND: Lung resistance-related protein (LRP), the major vault protein in humans, is sometimes overexpressed in multidrug-resistant cells. Because cells transfected with the LRP gene did not express the multidrug-resistant phenotype, we investigated whether LRP is involved in multidrug resistance. METHODS: SW-620 cells, a human colon carcinoma cell line, alone or transfected with an expression vector carrying a LRP-specific ribozyme or with an empty vector, were treated with sodium butyrate to induce differentiation. Expression of P-glycoprotein, multidrug resistance protein, and LRP in the cells was examined by northern and western blotting, and the efflux of doxorubicin in the cells or isolated nuclei was examined by fluorescence microscopy. RESULTS: A 2-week treatment with sodium butyrate induced LRP and conferred resistance to doxorubicin, vincristine, etoposide, gramicidin D, and paclitaxel (Taxol) in SW-620 cells. Insertion of either of two LRP-specific ribozymes into SW-620 cells inhibited these activities. Levels of drugs accumulating in the cells were not decreased by sodium butyrate, suggesting that the adenosine triphosphate-binding cassette transporter is not involved in sodium butyrate-induced multidrug resistance. Doxorubicin was mainly located in the nuclei of untreated cells and in the cytoplasm of sodium butyrate-treated cells. Isolated nuclei from untreated cells or sodium butyrate-treated cells incubated with anti-LRP polyclonal antibodies contained more doxorubicin than the nuclei of sodium butyrate-treated cells alone. Efflux of doxorubicin was greater from the nuclei of sodium butyrate-treated cells than the nuclei of untreated cells or of sodium butyrate-treated cells transfected with a LRP-specific ribozyme and was inhibited by an anti-LRP polyclonal antibody. CONCLUSIONS: LRP is involved in resistance to doxorubicin, vincristine, etoposide, paclitaxel, and gramicidin D and has an important role in the transport of doxorubicin from the nucleus to the cytoplasm.

Enhanced adenovirus transgene expression in malignant cells treated with the histone deacetylase inhibitor FR901228.

The presence of coxsackie and adenovirus receptor (CAR) and alpha(v) integrin on cell surfaces is required for efficient adenovirus infection. Treatment of cells with the histone deacetylase inhibitor FR901228 (depsipeptide) increased CAR and alpha(v) integrin RNA levels in six cancer cell lines. Sodium butyrate and trichostatin A, other histone deacetylase inhibitors, caused similar increases. Cells treated with FR901228 prior to infection had a 4-10-fold increase in transgene expression from a beta-galactosidase-expressing adenoviral vector. These studies suggest that FR901228 increases the efficiency of adenoviral transgene expression and may be useful in cancer gene therapy.

Suppression of metastasis by thymidine phosphorylase inhibitor.

We developed a novel inhibitor of thymidine phosphorylase (TP), 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride (TPI), that is about 1000-fold more active than 6-amino-5-chlorouracil, one of the most potent TP inhibitors. TPI inhibited the high chemotactic motility and basement membrane invasion of KB/TP cells, a TP-positive clone transfected with Rous sarcoma virus (RSV)/TP, to the levels seen in KB/CV cells, a control clone transfected with RSV. In nude mice, oral administration of TPI suppressed not only macroscopic liver metastases of highly metastatic KB/TP cells but also the level of human beta-globin as a molecular marker of micrometastases in the livers of the mice. These findings demonstrate that TP plays a key role in the invasiveness and metastasis of TP-expressing solid tumors and suggest that TPI might be a novel antimetastatic agent for blood-borne metastasis.

Construction of gene therapy vectors targeting thyroid cells: enhancement of activity and specificity with histone deacetylase inhibitors and agents modulating the cyclic adenosine 3',5'-monophosphate pathway and demonstration of activity in follicular and anaplastic thyroid carcinoma cells.

Thyroid carcinoma accounts for the majority of deaths from endocrine cancers. Although effective therapies exist for well differentiated tumors, the treatment options for poorly differentiated and anaplastic tumors are much less effective. In the present study we demonstrate that the thyroglobulin (Tg) promoter can be used to direct specific expression of either luciferase or thymidine kinase in thyroid cancer cells. Furthermore, using a putative enhancer element for the Tg gene, the activity of the Tg promoter in and its specificity for thyroid cells were enhanced. In transient transfectants or in stably transfected thyroid carcinoma cells, treatment with the histone deacetylase inhibitors, depsipeptide (FR9012228) and sodium butyrate, alone or in combination with 8-bromo-cAMP, resulted in further enhancement. In experiments in which the herpes simplex virus thymidine kinase (HSV-TK) gene was driven by the Tg promoter and the putative enhancer, HSV-TK expression and ganciclovir sensitivity were augmented. Similar results were obtained in two cell lines derived from a follicular thyroid carcinoma and in two anaplastic thyroid carcinoma cell lines. In summary, we report the construction of a suicide HSV-TK vector with preferential toxicity for thyroid cells. The results in anaplastic thyroid carcinoma cells suggest that it may be of use in the full spectrum of thyroid malignancies.

Low concentrations of the histone deacetylase inhibitor, depsipeptide (FR901228), increase expression of the Na(+)/I(-) symporter and iodine accumulation in poorly differentiated thyroid carcinoma cells.

Thyroid carcinoma accounts for the majority of deaths from endocrine cancers. A major cause of treatment failure is the inability to trap iodine. Chemotherapeutic agents with differentiating properties have been tried in an attempt to increase iodine uptake. We examined the ability of the novel histone deacetylase (HDAC) inhibitor, depsipeptide (FR901228), to modulate the expression of thyroid-specific genes. Four cell lines, two derived from follicular thyroid carcinomas (FTC 133 and FTC 236) and two derived from anaplastic thyroid carcinomas (SW-1736 and KAT-4) were used. In these four cell lines, a very low concentration of depsipeptide (1 ng/mL) increased histone acetylation and expression of both thyroglobulin and the Na(+)/I(-) symporter messenger RNAs. After 3 days, messenger RNA levels approached those of a normal thyroid control. Depsipeptide induced increases in (125)I accumulation indicated that a functional Na(+)/I(-) symporter protein was induced. Transient transfections indicate that the effects are mediated at least in part by a trans-activating factor. These in vitro results suggest that depsipeptide or other histone deacetylase inhibitors might be used clinically in thyroid carcinomas that are unable to trap iodine as an adjunct to radioiodine therapy.

Increased sensitivity to vincristine of MDR cells by the leukotriene D4 receptor antagonist, ONO-1078.

The leukotriene D4 (LTD4) receptor antagonist, 4-oxo-8-[p-(4-phenylbutyloxy)benzoylamino]-2-(tetrazol-5-yl) -4H-1-benzopyran hemihydrate (ONO-1078) is used for the treatment of allergic asthma and other immediate hypersensitivity diseases. We examined the effect of ONO-1078 on the sensitivity to vincristine (VCR) of MRP overexpressing multidrug-resistant CV60 and its parental drug-sensitive KB-3-1 cell lines. The sensitivity to VCR of KB-3-1 and CV60 cells was increased 13- and 15-fold, respectively, by ONO-1078 at the maximum non-toxic concentration (100 microM). The VCR sensitivity of multidrug-resistant KB-C2 cells that overexpressed P-gp was increased 2.6-fold by ONO-1078. The accumulation of VCR in KB-3-1, CV60 and KB-C2 cells was significantly increased by ONO-1078. The efflux of VCR from KB-3-1 cells was not inhibited, but that from CV60 cells was enhanced compared with that from KB-3-1 cells and was partially inhibited by ONO-1078. ONO-1078 competitively inhibited the ATP-dependent [3H]LTC4 uptake in membrane vesicles isolated from CV60 cells. These findings suggest that ONO-1078 inhibits the transporting activity of MRP and that ONO-1078 increases the sensitivity to VCR of KB-3-1 cells by increasing the VCR uptake in the cells.

Cardiac myxoma: morphologic, histochemical, and tissue culture studies.

Twenty-one cases of cardiac myxoma exhibiting a variety of histologic findings were investigated by light and electron microscopy, tissue culture, and immunohistochemical studies for factor VIII-related antigen (FVIII-RA) and Ulex europaeus 1 (UEA-1) lectin. The cardiac myxoma cells revealed variable cellular arrangements, and some tumor cells revealed vascular-like channels. Immunohistochemically, FVIII-RA was found only in the endothelial-like cells covering the vascular-like channels or slits, whereas UEA-1 lectin reacted not only with myxoma cells having luminal structures or network-like arrangements but also with single cells. On electron microscopy, some myxoma cells revealed differentiation into cells forming vascular structures. In tissue culture, the tumor cells were polygonal and proliferated with extensions of the cytoplasmic processes. Arrangements suggesting vascular channels or slits were not observed. In a coculture of tumor cells and blood clot, the tumor cells covered the surface of the clot. However, angiogenesis was not observed in the tissue culture study. The results of our studies were inconclusive regarding the histogenesis of cardiac myxomas, but it was considered that cardiac myxoma is a neoplasm arising from mesenchymal cells with vasoformative characteristics.

Primary choriocarcinoma of the lung.

Choriocarcinoma was found in the lung of a 34-year-old woman. Examination of the patient's entire body, especially the genital tract, failed to disclose foci of choriocarcinoma other than that in the right lung. After surgery, the levels of human chorionic gonadotropin in the blood and the urine fell. It was concluded that the choriocarcinoma of the lung was, in fact, the primary tumor. The genesis of choriocarcinoma was also studied in ten patients who died after delivery or abortion. Autopsy disclosed trophoblasts in the pulmonary arteries in nine of these ten patients. These findings suggest that primary choriocarcinoma in women is due to pulmonary embolism caused by trophoblasts at the time of abortion or delivery.

[Mechanisms for resistance to anticancer agents and the reversal of the resistance].

MDR results from overexpression of P-glycoprotein (Pgp) and multidrug resistance protein (MRP or MRP1) that function as ATP-dependent efflux pumps. Lung resistance related protein (LRP) is also supposed to be involved in MDR. The human canalicular multispecific organic anion transporter (cMOAT) gene that is responsible for the defects in Dubin-Johnson syndrome was isolated. cMOAT is homologous to MRP1 and supposed to be involved in drug resistance. Human cMOAT cDNA transfected LLC-PK1 cells, LLC/cMOAT-1, have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxycamptothecin (SN-38), and cisplatin. The multidrug resistance (MDR)-reversing agents, cyclosporin A (CsA) and PAK-104P, almost completely reversed the resistance to VCR, SN-38 and cisplatin of LLC/cMOAT-1 cells by interacting with the substrate binding site of cMOAT. Treatment of human colorectal carcinoma SW-620 cells with sodium butyrate(NaB) induced LRP in the cells and conferred resistance to Adrianycin(ADM), VCR, VP-16, gramicidin D and taxol. Two LRP-specific ribozymes inhibited the NaB-induced expression of LRP in SW-620 cells and almost completely abolished their acquisition of the MDR phenotype. The accumulation of ADM, VCR and taxol was not decreased in NaB-treated cells, suggesting that ATP-binding cassette transporters are not involved in the MDR of NaB-treated cells. ADM was mainly located in the nuclei of untreated and the cytoplasm of NaB-treated cells. The accumulation level of ADM in the nuclei isolated from untreated cells or those from treated cells in the presence of anti-LRP polyclonal antibody was higher than that from treated cells in the absence of the antibody. Efflux of ADM from nuclei isolated from NaB-treated cells was enhanced compared with those from untreated cells and NaB-treated cells transfected with a LRP-specific ribozyme. The polyclonal antibody against LRP inhibited the enhanced efflux of ADM from nuclei isolated from NaB-treated cells. These findings indicate that LRP is involved in resistance to ADM, VCR, VP-16, taxol and gramicidin D, and has an important role in the transport of ADM from the nucleus to the cytoplasm.

Superficial intimal injury of the rabbit carotid artery induced by distilled water.

The study of mechanical injury to the aortic endothelium in experimental animals is important in understanding the pathologic processes in atherogenesis. In this investigation distilled water was used to produced superficial injury to the rabbit carotid artery. Sterilized distilled water was injected into a temporarily isolated segment of rabbit carotid artery measuring 0.5 cm in length. After 4 min blood flow was reestablished by removal of the isolating ligatures. The carotid arteries were examined at time intervals of 5 min, 24 h, 48 h, 1 month, 3 months and 6 months after injury. Five min after injury, the carotid endothelial cells were almost completely removed but no medical injury was present. After 24 and 48 h, a few platelets were adherent to the denuded intimal surface. After 1 month, 3 months and 6 months the injured surface showed a slight intimal thickening consisting of modified smooth muscle cells. Our experimental findings suggested that the extent of the injured area is more important in the repair process than its depth.

Calcification of matrix vesicles in cardiac myxoma.

The mechanism of calcification in cardiac myxoma, using histologic, ultrastructural and potassium pyroantimonate technique was demonstrated. There are a large number of extracellular membrane-invested structure measuring 100-700 millimicrons in cardiac myxoma. These structures are similar to the matrix vesicles observed in skeletal tissue and atherosclerotic foci. Potassium pyroantimonate reacts with these vesicular structures and degenerated myxoma cells. It was thought that calcification in cardiac myxoma occurs by the same mechanism as calcification in atherosclerosis.

[The influence of green tea upon the improvement of iron deficiency anemia with pregnancy treated by sodium ferrous citrate].

Green tea is indispensable to our everyday life. In Japan it has long been common knowledge that the ingestion of green tea should be avoided before and after the intake of iron preparations. There have recently been some reports, however, that deny the effect of green tea on iron preparations. A study was conducted on pregnant patients with anemia, using sodium ferrous citrate (Ferromia). The drug was administered to a group of patients taking green tea and a group taking water. Our results can be summarized as follows. 1. Hemoglobin, serum iron and total iron binding capacity were markedly improved after the administration of the iron preparation, and there was no difference between these parameters in the two groups of patients. 2. There was a tendency for patients with hypochromia to show a more marked improvement in hemoglobin in both groups. 3. Anemia cured in 96.7% of patients in the green tea group and in 93.4% of patients in the water group after the oral administration of the iron preparation. 4. The incidence of side effects stood at 18.3% for the green tea group and 21.9% for the water group, there being no significant difference. No serious side effects were elicited in the present study.

The surface lining cells of cardiac myxoma. Light, electron microscopic and immunohistochemical observation.

Morphological, enzymatic and electron microscopic studies have been performed on the surface covering cells of cardiac myxoma (CM) in 4 clinical cases. The superficial layer of CM is composed of mono- or poly-layers of polygonal cells and the superficial cells are connected with the tumor cells within CM. Electron microscopic picture showed many microvilli on the surface of the superficial cells, and the neighboring cells extend extrusions one to another connecting with desmosomes. Formation of incomplete basal membrane was observed in the underlying cells. These cells were morphologically similar to those of the tumor cells in CM. Polygonal superficial cells were shown by scanning electron microscope, and there was no consistent arrangement, which was different from the vascular endothelial cells. Factor VIII-related antigen was proved in the tumor cells having vascular like space in CM, suggesting differentiation of the tumor cells to the vascular endothelial cells. Factor VIII-related antigen was negative in the superficial covering cells. It may be said that the superficial covering cells of CM are premature tumor cells having no character as endothelial cells.

[Malignant fibrous histiocytoma of the scrotum].

A 72-year-old man was hospitalized for a gradually enlarging left scrotal mass. Physical examination revealed a 3 X 4 cm. rock hard non-tender mass fixed to adacent tissue. The tumor invaded the skin of scrotum and showed ulcer formation. Surgical therapy was performed under the diagnosis of left scrotic tumor. The resected mass was examined histologically, and the pathologic diagnosis was of malignant fibrous histiocytoma of the left scrotum was made.

Nodular regenerative hyperplasia of the liver in a patient with multiple myeloma and systemic amyloidosis.

A 68-year-old man with multiple myeloma and systemic amyloidosis also had nodular regenerative hyperplasia (NRH) of the liver. This unusual case is discussed and the association of NRH with various other systemic disorders are reviewed.

Patients with homocystinuria: high metal concentrations in hair, blood and urine.

Patients with homocystinuria excrete a large amount of metal in their urine. Homocysteine similar to penicillamine, administration to methylmercury treated rats resulted in a large amount of urinary methylmercury excretion. These results suggested that the total metal amounts in the whole body of patients with homocystinuria might be decreased. However, actually metal concentrations in hair and plasma of these patients were higher than those of normal controls. High plasma and hair metal levels are not accounted for in patients with homocystinuria. The physiological metal excretory mechanism in which small amounts of metals bind to the small, plasma molecular substances filter through the kidney and emerge in the urine is necessary for reconfirmation. Strongly perturbed metal metabolism exists in the patients with homocystinuria.

Reversal of LRP-associated drug resistance in colon carcinoma SW-620 cells.

Resistance to multiple drugs is mediated by lung resistance-related protein (LRP) as well as P-glycoprotein (P-gp) and multidrug resistance protein (MRP). The levels of expression of LRP mRNA and LRP in a human colon carcinoma cell line, SW-620, were increased by the differentiation-inducing agent, sodium butyrate (NaB). Treatment of SW-620 cells with NaB for 2 weeks conferred resistance to adriamycin (ADM) and VP-16. The resistance was almost completely reversed by PAK-104P, a pyridine analog, but not by cepharanthine. ADM accumulated mainly in the nuclei of SW-620 cells not treated with NaB and in the cytoplasm of SW-620 cells treated with NaB. When the NaB-treated SW-620 cells were incubated with ADM in the presence of PAK-104P, the accumulation of ADM in nuclei was substantially increased. Isolated nuclei from untreated cells accumulated more ADM than nuclei from NaB-treated cells. Efflux of ADM from the nuclei isolated from NaB-treated cells was enhanced. PAK-104P and an antibody against LRP increased the accumulation of ADM in the isolated nuclei from NaB-treated cells, and inhibited the enhanced efflux of ADM from the nuclei. These findings suggest that at least in part, PAK-104P reverses LRP-mediated drug resistance by inhibiting the efflux of ADM from nuclei. PAK-104P may be useful for reversing MDR in tumors that overexpress LRP.

The expression of multidrug resistance protein in human gastrointestinal tract carcinomas.

BACKGROUND: Multidrug resistance protein (MRP) is a membrane phosphoglycoprotein with an Mr of 190,000 that is involved in the non-P-glycoprotein mediated multidrug resistance of human tumor cells. The aim of this study was to determine the clinicopathologic relevance of MRP expression in human gastrointestinal tract carcinomas. METHODS: The authors prepared a rabbit antiserum against MRP that does not cross-react with P-glycoprotein and retrospectively examined the expression of MRP in 86 squamous cell carcinomas of the esophagus, 103 adenocarcinomas of the stomach, and 139 colorectal adenocarcinomas by immunohistochemistry. None of the patients in this study had received prior chemotherapy. RESULTS: The proportion of MRP positive samples in the squamous cell carcinomas of the esophagus (62.8%, 54 of 86) was significantly higher than that in the adenocarcinomas of the stomach (34.1%, 35 of 103) and the colorectal adenocarcinomas (40.3%, 56 of 139) (P <0.01). The proportion of MRP positivity in the well-differentiated carcinomas was significantly higher than that in moderately or poorly differentiated carcinomas. MRP expression was independent of gender, lymph node metastasis, and tumor progression. CONCLUSIONS: These data indicate that the expression of MRP is correlated with the differentiation of carcinoma cells in the gastrointestinal tract and may be involved in the intrinsic drug resistance of well-differentiated squamous cell carcinoma of the esophagus.

Prevention of hypoxia-induced apoptosis by the angiogenic factor thymidine phosphorylase.

The angiogenic factor platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is expressed at higher levels in a wide variety of solid tumors compared to adjacent normal tissues. Patients with PD-ECGF/TP-positive colon and esophageal tumors have a poorer prognosis than those with negative tumors. The expression of PD-ECGF/TP is a prognostic factor independent of microvessel density suggesting that TP has effects on tumor progression independent of its angiogenic activity. Evidence that hypoxia and apoptosis affect tumor growth prompted us to determine whether increased expression of PD-ECGF/TP prevents apoptosis induced by hypoxia. KB/TP cells transfected with a PD-ECGF/TP cDNA were resistant to hypoxia-induced apoptosis. Among the degradation products of thymidine produced by PD-ECGF/TP, 2-deoxy-D-ribose and thymine partially prevented hypoxia-induced apoptosis. The ability of 1 microM 2-deoxy-D-ribose in combination with the same concentration of thymine to prevent hypoxia-induced apoptosis was similar to that of the overexpressed TP in KB cells. A concentration of 1 microM 2-deoxy-L-ribose abrogated the effects of these degradation products of thymidine. These findings suggested that TP can confer resistance to apoptosis induced by hypoxia and the degradation products of thymidine are involved in this resistance. Expression of PD-ECGF/TP may play an important role in the progression of solid tumors, and inhibitors of TP and analogs of the degradation products of thymidine may suppress the growth of tumors by promoting apoptosis.

[Approach to the diagnostic colposcopy--a scoring method of colposcopic pattern].

In order to arrive at a final diagnosis of early cervical cancer by colposcopy alone, a colposcopic scoring system was applied to 172 patients with the disease. Each colposcopic pattern was scored from one point (for white epithelium) to 9 (for invasive cancer). The results obtained were as follows; 1) With these scoring criteria, stage 0 cancers were restricted to below 10 points and were characterized colposcopically by white epithelia with gland openings 2) In stage I-a lesion, atypical vessels, punctuation by irregular arrangement of the dots and concentration of abnormal gland openings were usually observed, and these findings were combined with each other to show a more complicated colposcopic pattern reflected in 11 to 18 points of the score 3) Frank invasions were indicated by 19 points or more, and punctuations or mosaics were seldom found in this stage, although the early "I-b" cancer was difficult to distinguish from I-a with this method 4) The diagnoses from this scoring system were identical with the final diagnoses confirmed by surgical methods in 66.7% of stage 0, 59.4% of I-a and 76.5% of I-b.