Mechanism of specific nuclear transport of adriamycin: the mode of nuclear translocation of adriamycin-proteasome complex.

Adriamycin (ADM), an anthracycline anticancer agent, is selectively stored in the nuclei of a variety of proliferating cells, but the precise mechanism of specific nuclear transport of ADM is not well known. Recently, we demonstrated that ADM shows high binding affinity to the cytoplasmic proteasomes of L1210 mouse leukemia cells and that taken up ADM by the cells selectively binds to proteasomes. Nuclear targeting of proteasome in proliferating cells may be mediated by the nuclear localization signals that are found in several of the alpha-type subunits of the 20S proteasome. To confirm nuclear transport of the ADM-proteasome complex, we synthesized a photoactive ADM analogue, N-(p-azidohenzoyl)-ADM, and generated a photoaffinity-labeled proteasome complex. The 26S proteasome purified from the cytosol of L1210 cells had a high affinity to N-(p-azidobenzoyl)-ADM. SDS-PAGE analysis of the photoaffinity-labeled proteasome showed that low molecular weight bands (approximately 21-31 kDa) of 20S proteasome had the highest photoaffinity. The photoaffinity-labeled proteasome was distributed in the cytoplasm and nuclei of digitonin-permeabilized L1210 and B-16 mouse melanoma cells in the presence of the cytosolic fraction and ATP. The rate of nuclear translocation of the proteasome was low in the absence of ATP. These results suggest that the proteasome is a specific translocator of ADM from the cytoplasm to the nucleus and that 20S proteasome components are the dominant ADM-binding sites. The nuclear transport of ADM-proteasome complex is regulated by an ATP-dependent nuclear pore-mediated mechanism.

Differences in intracellular sites of action of Adriamycin in neoplastic and normal differentiated cells.

PURPOSE: This study was performed to clarify the intracellular specificity of the differential cytotoxic effects of Adriamycin (ADM) on neoplastic and normal cells. METHODS: The mouse lymphocytic leukemia cell line L1210 and pig kidney proximal tubular epithelial cell line LLC-PK1 were used as neoplastic and normal cells, respectively. These cells were treated with various concentrations of ADM for 24 h and toxicological parameters were determined. RESULTS: ADM (0.1-10 microM) significantly down-regulated cell growth rate and [3H]thymidine incorporation into DNA in the log phase, and at concentrations of more than 1 microM reduced the viability of both cell lines. Lipid peroxidation was increased at 1 microM ADM in L1210 cells and at 10 microM ADM in LLC-PK1 cells. The microsomal and nuclear fractions of both cell lines showed approximately the same level of ADM-induced superoxide anion (O2-) production, while the mitochondrial fraction of differentiated LLC-PK1 cells produced the highest levels of O2-. Differentiated LLC-PK1 cells showed the highest mitochondrial NADH-cytochrome c reductase activity. L1210 cells showed lower mitochondrial activities of enzymes involved in scavenging of reactive oxygen species, such as superoxide dismutase, glutathione peroxidase and catalase, than the other cells. CONCLUSIONS: These results suggest that ADM exerts cytostatic effects on neoplastic and normal undifferentiated cells through the inhibition of DNA synthesis by DNA intercalation, and cytotoxic effects on neoplastic cells through the accumulation of reactive oxygen species resulting from low scavenger enzyme activities. The cytotoxic effects on normal differentiated cells may be related to the high levels of production of reactive oxygen species due to high mitochondrial NADH-cytochrome c reductase activity.

Differential toxic effects of gentamicin on cultured renal epithelial cells (LLC-PK1) on application to the brush border membrane or the basolateral membrane.

Aminoglycoside antibiotics are generally accepted to accumulate in renal proximal tubule cells from the luminal surface and show toxic effects on the cells. The binding affinity and membrane permeability of aminoglycoside antibiotics are different at the brush border membrane (BBM) and the basolateral membrane (BLM) of proximal tubule cells. This study was performed, therefore, to investigate the differential effects of the aminoglycoside antibiotic gentamicin (GM) on cultured LLC-PK1 cells, a pig kidney proximal epithelial cell line, after addition to the BBM or the BLM side. LLC-PK1 cells were cultured on microporous membranes until forming confluent monolayers, and then GM was added to either the BBM or the BLM side. GM caused release of enzymes from the organelles, with a higher level of release observed following addition to the BBM side than that to the BLM side. Patterns of [3H]GM uptake by the cells differed in a manner dependent on whether it was added to the BBM or the BLM side. That is, the cellular uptake from the BBM side increased with incubation time, while that from the BLM side showed rapid saturation. These results suggested that aminoglycoside antibiotics show differential effects on cultured proximal epithelial cells and have differential patterns of cellular uptake when added to the BBM or the BLM side.

Roles of oxygen radical production and lipid peroxidation in the cytotoxicity of cephaloridine on cultured renal epithelial cells (LLC-PK1).

To clarify the mechanism of cephalosporin nephrotoxicity, the cytotoxic effects of cephaloridine (CER), a nephrotoxic cephalosporin antibiotic, on the pig kidney proximal tubular epithelial cell line (LLC-PK1) were studied in culture. CER increased the content of hydrogen peroxide and decreased the activity of catalase in the treated cells, followed by an increase in the content of lipid peroxide and decreases in both glutathione peroxidase activity and in the non-protein sulfhydryl content. The levels of NADPH-dependent hydrogen peroxide and superoxide anion production by microsomes prepared from LLC-PK1 cells, and by NADPH-cytochrome P-450 reductase purified from the rat renal cortex were significantly increased by paraquat. The production of these molecules was antagonized by p-chloromer-curibenzoate, an inhibitor of NADPH-cytochrome P-450 reductase. On the other hand, CER did not significantly affect the production of hydrogen peroxide or superoxide anions. These results suggested that the cytotoxic effect of CER on cultured LLC-PK1 cells was due to the increases in hydrogen peroxide and lipid peroxide levels and not microsomal oxygen radical production, and that the mechanism of this cytotoxicity is very different from that of paraquat which induces microsomal oxygen radical production.

Cephaloridine-induced inhibition of cytochrome c oxidase activity in the mitochondria of cultured renal epithelial cells (LLC-PK(1)) as a possible mechanism of its nephrotoxicity.

To clarify the mechanism of cephalosporin nephrotoxicity, the effects of cephaloridine (CLD), a nephrotoxic cephalosporin antibiotic, on the mitochondria of the pig kidney proximal tubular epithelial cell line LLC-PK(1) were studied in culture. The activity of cytochrome c oxidase in the mitochondria of LLC-PK(1) cells was significantly decreased from 9 h after addition of 1.0 mM CLD to the cultured cells. These effects were dose-dependent and accompanied with a significant decrease in the ATP content in the cells, followed by marked morphological changes in the mitochondria. These alterations were observed in the treated cells before the increase in lipid peroxidation. The activities of NADH-cytochrome c reductase and succinate dehydrogenase in the mitochondria and NADPH-cytochrome P450 reductase, NADH-cytochrome b(5) reductase, and 7-ethoxycoumarin O-deethylase in the microsomes of the treated cells were not affected. Superoxide anion production by the mitochondria prepared from LLC-PK(1) cells or NADH-cytochrome c reductase was not affected by addition of CLD (1-10 mM), but adriamycin (0.1 mM) or paraquat (0.1 mM) significantly increased the superoxide anion production. These results suggested that the primary action of CLD is inhibition of cytochrome c oxidase activity in the mitochondrial electron transport chain, which decreases intracellular ATP content in renal tubular epithelial cells and that these effects of CLD are followed by increased lipid peroxidation and cellular injury.

In situ photoaffinity labeling of proteasome with photoactive adriamycin analogue.

An intracellular adriamycin (ADM)-binding protein purified from the cytosol of L1210 mouse lymphocytic leukemia cells had a molecular weight of 700-1500 kDa and hydrolyzed Suc-LLVY-MCA. When L1210 cells were incubated with a photoactive ADM analogue, N-(p-azidobenzoyl)-adriamycin (NAB-ADM), most of the NAB-ADM was found to localize in the nuclei. In situ photoaffinity labeling of L1210 cells with NAB-ADM resulted in low protease activity in the cytosol and nuclear extracts and the cells showed selective photoincorporation of NAB-ADM into the proteasome. These results suggest that the proteasome is a translocator of ADM from the cytoplasm to the nucleus and might therefore become a new candidate for cancer chemotherapy.

Role of hypothermia induced by tumor necrosis factor on apoptosis and function of inflammatory neutrophils in mice.

Changes in body temperature and cell infiltration, mediated by cytokines including tumor necrosis factor-alpha (TNF-alpha), occur during inflammation, but a role of body temperature on inflammatory responses remains obscure. Intraperitoneal injection of 10% casein to mice resulted in transient hypothermia followed by neutrophil accumulation in peritoneal cavities. Peritoneal TNF-alpha was rapidly raised, and pretreatment of mice with an anti-TNF-alpha antibody promoted temperature restoration and partially inhibited neutrophil accumulation. To investigate direct effects of body temperature on neutrophils, peritoneal or peripheral blood neutrophils were cultured at 35 degrees C or 37 degrees C with or without recombinant murine TNF-alpha (100 ng/ml) or a protein synthesis inhibitor cycloheximide (1 microg/ml). Significant inhibition of spontaneous and TNF-alpha-induced apoptosis was obtained at 35 degrees C compared with 37 degrees C, an effect that was not altered by the addition of cycloheximide. Moreover, phagocytic ability of peritoneal neutrophils was significantly enhanced by incubating them at the lower temperature. These results indicate that mild hypothermia induced by endogenous TNF-alpha has enhancing roles on neutrophil survival and function during peritoneal inflammation.

Implanted tumor growth is suppressed and survival is prolonged in sixty percent of food-restricted mice.

To examine the effect of food restriction on immune functions in the tumor-bearing state, mice were divided into a control group (fed 5.0 g diet/d; 71 kJ/d) and a 60% food-restricted group (fed 3.0 g diet/d; 43 kJ/d) at 8-wk of age, and 4 wk later, L1210 tumor cells were inoculated intradermally. In the food-restricted mice, tumor growth was significantly suppressed, and mean survival time after the tumor inoculation was prolonged (P < 0.05). The plasma concentrations of two antitumor cytokines, interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), were greater in the food-restricted group before tumor inoculation (P < 0. 05). Furthermore, the food-restricted mice had significantly higher plasma levels of IFN-gamma and TNF-alpha after tumor inoculation, although the treatment significantly increased these cytokine levels in both groups. Splenic natural killer cell cytotoxicity was also higher in the tumor-bearing food-restricted mice than in controls (P < 0.05). Food-restricted mice have strong antitumor immunity, and as a result, tumor growth is suppressed and survival time is prolonged in these mice.

Proteasome is a carrier to translocate doxorubicin from cytoplasm into nucleus.

When an effective concentration of doxorubicin (DXR) was added into L1210 of a mouse leukemia cell line, DXR was rapidly distributed much more in the nuclei than in the other organelle within a few minutes. A [14C]DXR-binding fraction was obtained from the cytosol prepared from L1210 cells. The fraction was adsorbed to hydroxylapatite matrix and eluted from the matrix by 50-150 mM potassium phosphate buffer. The fraction showed high DXR-binding and Suc-Leu-Leu-Val-Tyr-MCA-degrading activity. The binding of [14C]DXR was inhibited by unlabeled DXR. Gel chromatography of the fraction with Sephacryl S-300 separated two fractions of high molecular weight (Peak I, approx. 750 kDa) and low molecular weight (Peak II). Peak I showed proteolytic activity. [14C]DXR-binding Peak I had much higher affinity to DNA-cellulose than [14C]DXR-binding Peak II. [14C]DXR-Peak I complex also was retained into the nuclei isolated from L1210 cells, temperature-dependently. These results suggest that a specific carrier to translocate DXR from cytoplasm into nucleus exists in L1210 cell and the carrier is proteasome.

Mechanism of toxic action of fluoride in dental fluorosis: whether trimeric G proteins participate in the disturbance of intracellular transport of secretory ameloblast exposed to fluoride.

In enamel fluorosis model rats treated with sodium fluoride, secretory ameloblasts of incisor tooth germs exhibited disruption of intracellular trafficking. We examined whether heterotrimeric G proteins participated in the disruption of vesicular trafficking of the secretory ameloblast exposed to fluoride, using immunoblotting and pertussis toxin (IAP)-induced adenosyl diphosphate (ADP)-ribosylation for membrane fractions of the cell. Immunoblotting of crude membranes, post supernatants of the ameloblast, with anti-G(alpha i3/alpha o) and anti-G(alpha s) antibodies showed that Gi3 or Go proteins existed in the secretory ameloblast, but Gs protein did not. Immunoblotting of the subcellular membrane fractions indicated that the Gi3 or Go proteins were located in the Golgi membrane, but were not in the rough endoplasmic reticulum (rER) membrane. Autoradiograph of IAP-induced ADP-ribosylation, however, showed the existence of IAP-sensitive G proteins both in rER and Golgi membranes. Fluoride treatment decreased the G proteins bound to both membranes. These findings indicate that different G proteins, both of which are IAP-sensitive, are present in the rER and Golgi apparatus, and suggest that these G proteins participate in the disturbance of intracellular transport of the secretory ameloblast exposed to fluoride.

Involvement of arachidonic acid metabolites in acute inflammation: detection of 6-keto-PGF1 alpha, thromboxane B2 and PGD2 in rat pleurisy induced by phorbol myristate acetate.

Rat pleurisy was induced by intrapleural injection of phorbol myristate acetate (PMA), a known tumor promotor and a component of croton oil. Pleural fluids at 30 min and 1 hr after PMA-injection were collected and arachidonic acid metabolites in the fluids were measured by RIA or bioassay after fractionation through reversed phase HPLC using an ODS column. The major metabolites found in the pleural fluid were 6-keto-PGF1 alpha, TXB2 and PGD2, with a small amount of PGE2. Pretreatment with 10 mg/kg indomethacin suppressed the pleural fluid accumulation and also reduced the amount of the above metabolites to the basal levels. Treatment with OKY-046, a novel thromboxane synthetase inhibitor, reduced the level of TXB2 completely, but had no effect on those of 6-keto-PGF1 alpha and PGD2, and it had no effect on pleural fluid accumulation either. The results may indicate that PGI2 plays a role for the vascular permeability increase in the early phase of pleurisy.