Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death.

The spatial distribution of radiation-induced DNA breaks within the cell nucleus depends on radiation quality in terms of energy deposition pattern. It is generally assumed that the higher the radiation linear energy transfer (LET), the greater the DNA damage complexity. Using a combined experimental and theoretical approach, we examined the phosphorylation-dephosphorylation kinetics of radiation-induced γ-H2AX foci, size distribution and 3D focus morphology, and the relationship between DNA damage and cellular end points (i.e., cell killing and lethal mutations) after exposure to gamma rays, protons, carbon ions and alpha particles. Our results showed that the maximum number of foci are reached 30 min postirradiation for all radiation types. However, the number of foci after 0.5 Gy of each radiation type was different with gamma rays, protons, carbon ions and alpha particles inducing 12.64 ± 0.25, 10.11 ± 0.40, 8.84 ± 0.56 and 4.80 ± 0.35 foci, respectively, which indicated a clear influence of the track structure and fluence on the numbers of foci induced after a dose of 0.5 Gy for each radiation type. The γ-H2AX foci persistence was also dependent on radiation quality, i.e., the higher the LET, the longer the foci persisted in the cell nucleus. The γ-H2AX time course was compared with cell killing and lethal mutation and the results highlighted a correlation between cellular end points and the duration of γ-H2AX foci persistence. A model was developed to evaluate the probability that multiple DSBs reside in the same gamma-ray focus and such probability was found to be negligible for doses lower than 1 Gy. Our model provides evidence that the DSBs inside complex foci, such as those induced by alpha particles, are not processed independently or with the same time constant. The combination of experimental, theoretical and simulation data supports the hypothesis of an interdependent processing of closely associated DSBs, possibly associated with a diminished correct repair capability, which affects cell killing and lethal mutation.

TM9SF4 is a novel V-ATPase-interacting protein that modulates tumor pH alterations associated with drug resistance and invasiveness of colon cancer cells.

An inverted pH gradient across the cell membranes is a typical feature of malignant cancer cells that are characterized by extracellular acidosis and cytosol alkalization. These dysregulations are able to create a unique milieu that favors tumor progression, metastasis and chemo/immune-resistance traits of solid tumors. A key event mediating tumor cell pH alterations is an aberrant activation of ion channels and proton pumps such as (H+)-vacuolar-ATPase (V-ATPase). TM9SF4 is a poorly characterized transmembrane protein that we have recently shown to be related to cannibal behavior of metastatic melanoma cells. Here, we demonstrate that TM9SF4 represents a novel V-ATPase-associated protein involved in V-ATPase activation. We have observed in HCT116 and SW480 colon cancer cell lines that TM9SF4 interacts with the ATP6V1H subunit of the V-ATPase V1 sector. Suppression of TM9SF4 with small interfering RNAs strongly reduces assembly of V-ATPase V0/V1 sectors, thus reversing tumor pH gradient with a decrease of cytosolic pH, alkalization of intracellular vesicles and a reduction of extracellular acidity. Such effects are associated with a significant inhibition of the invasive behavior of colon cancer cells and with an increased sensitivity to the cytotoxic effects of 5-fluorouracil. Our study shows for the first time the important role of TM9SF4 in the aberrant constitutive activation of the V-ATPase, and the development of a malignant phenotype, supporting the potential use of TM9SF4 as a target for future anticancer therapies.

The thiazole derivative CPTH6 impairs autophagy.

We have previously demonstrated that the thiazole derivative 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) induces apoptosis and cell cycle arrest in human leukemia cells. The aim of this study was to evaluate whether CPTH6 is able to affect autophagy. By using several human tumor cell lines with different origins we demonstrated that CPTH6 treatment induced, in a dose-dependent manner, a significant increase in autophagic features, as imaged by electron microscopy, immunoblotting analysis of membrane-bound form of microtubule-associated protein 1 light chain 3 (LC3B-II) levels and by appearance of typical LC3B-II-associated autophagosomal puncta. To gain insights into the molecular mechanisms of elevated markers of autophagy induced by CPTH6 treatment, we silenced the expression of several proteins acting at different steps of autophagy. We found that the effect of CPTH6 on autophagy developed through a noncanonical mechanism that did not require beclin-1-dependent nucleation, but involved Atg-7-mediated elongation of autophagosomal membranes. Strikingly, a combined treatment of CPTH6 with late-stage autophagy inhibitors, such as chloroquine and bafilomycin A1, demonstrates that under basal condition CPTH6 reduces autophagosome turnover through an impairment of their degradation pathway, rather than enhancing autophagosome formation, as confirmed by immunofluorescence experiments. According to these results, CPTH6-induced enhancement of autophagy substrate p62 and NBR1 protein levels confirms a blockage of autophagic cargo degradation. In addition, CPTH6 inhibited autophagosome maturation and compounds having high structural similarities with CPTH6 produced similar effects on the autophagic pathway. Finally, the evidence that CPTH6 treatment decreased α-tubulin acetylation and failed to increase autophagic markers in cells in which acetyltransferase ATAT1 expression was silenced indicates a possible role of α-tubulin acetylation in CPTH6-induced alteration in autophagy. Overall, CPTH6 could be a valuable agent for the treatment of cancer and should be further studied as a possible antineoplastic agent.

Autophagy: Molecular mechanisms and their implications for anticancer therapies.

Autophagy is a catabolic process whereby cells maintain homeostasis by eliminating unnecessary proteins and damaged organelles. It may be triggered under physiological conditions, such as nutrient starvation, or in response to a variety of stress stimuli, such as exposure to radiations or cytotoxic compounds. Although autophagy is basically a protective mechanism that sustains cell survival under adverse conditions, it has been recently demonstrated that the induction of autophagic process may ultimately lead to cell death. As for the role of autophagy in cancer, it is still very controversial whether it suppresses tumorigenesis or provides cancer cells with a rescue mechanism under unfavourable conditions. Therefore, the dual role of autophagy in tumor progression and in the response of cancer cells to chemotherapeutic drugs is still open to debate. The first part of this review describes the cellular events occurring during the various phases of the autophagic process. Special attention has been given to the morphological aspects and the regulatory molecules involved in autophagic cell death. Specifically, we have focused on the proteins necessary for autophagosome formation, encoded by the ATG (AuTophaGy-related gene) gene family, and their role in the regulation of the process of autophagy. We also examined the effects of autophagy modulators on cell survival and cell death and discussed the recent efforts aimed at finding novel agents that activate or inhibit autophagy by targeting regulatory molecules of the complex autophagy pathways.

Proton pump inhibition induces autophagy as a survival mechanism following oxidative stress in human melanoma cells.

Proton pump inhibitors (PPI) target tumour acidic pH and have an antineoplastic effect in melanoma. The PPI esomeprazole (ESOM) kills melanoma cells through a caspase-dependent pathway involving cytosolic acidification and alkalinization of tumour pH. In this paper, we further investigated the mechanisms of ESOM-induced cell death in melanoma. ESOM rapidly induced accumulation of reactive oxygen species (ROS) through mitochondrial dysfunctions and involvement of NADPH oxidase. The ROS scavenger N-acetyl-L-cysteine (NAC) and inhibition of NADPH oxidase significantly reduced ESOM-induced cell death, consistent with inhibition of cytosolic acidification. Autophagy, a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, represents a defence mechanism in cancer cells under metabolic stress. ESOM induced the early accumulation of autophagosomes, at the same time reducing the autophagic flux, as observed by WB analysis of LC3-II accumulation and by fluorescence microscopy. Moreover, ESOM treatment decreased mammalian target of rapamycin signalling, as reduced phosphorylation of p70-S6K and 4-EBP1 was observed. Inhibition of autophagy by knockdown of Atg5 and Beclin-1 expression significantly increased ESOM cytotoxicity, suggesting a protective role for autophagy in ESOM-treated cells. The data presented suggest that autophagy represents an adaptive survival mechanism to overcome drug-induced cellular stress and cytotoxicity, including alteration of pH homeostasis mediated by proton pump inhibition.

Autophagy-mediated chemosensitizing effect of the plant alkaloid voacamine on multidrug resistant cells.

In our previous studies, voacamine, a bisindolic alkaloid extracted from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on multidrug resistant (MDR) human osteosarcoma cells (U-2 OS-R). Voacamine induced in resistant cells a significant increase of drug retention and intranuclear location which became comparable to those observed in the parental sensitive counterparts (U-2 OS-WT). In the present study, the cell survival analysis and the electron microscopic observations confirmed the evident cytotoxicity of DOX on MDR cells after pre-treatment with the plant extract. Moreover, an increase of the reactivity of P-glycoprotein (P-gp) with the monoclonal antibody UIC2, which recognizes an epitope of the drug transporter in its functional conformation, was revealed, demonstrating that voacamine is a substrate of P-gp, thus acting as a competitive antagonist of the cytotoxic agent. Moreover, to investigate if the enhancement of the cytotoxic effect induced by voacamine could be due to an apoptotic process, we carried out the analysis of cell morphology after Hoechst staining and the quantification of apoptosis by Annexin V-FITC assay. These evaluations showed a very low rate of apoptosis in U-2 OS-R cells treated with voacamine and DOX given in association. In addition, the combined treatment induced ultrastructural modifications suggestive of autophagic cell death. In particular, transmission electron microscopy observations revealed the presence of numerous lysosomes and the formation of a large number of autophagosomes containing residual digested material. In conclusion, these findings seem to indicate that voacamine is capable of enhancing the cytotoxic effect of DOX on MDR cells by favouring a lethal autophagic process.

Voacamine, an alkaloid extracted from Peschiera fuchsiaefolia, inhibits P-glycoprotein action in multidrug-resistant tumor cells.

Multidrug resistance (MDR) in tumor cells is generally associated with increased efflux of the cytotoxic compounds, due to the activation of mechanisms of intracellular transport and to the overexpression of surface proteins, such as P-glycoprotein (Pgp), which act as ATP-dependent molecular pumps. In a previous study, voacamine, a bisindolic alkaloid from Peschiera fuchsiaefolia, was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on resistant human osteosarcoma cells. The effects of voacamine on the cell survival and on accumulation of DOX were investigated on both the parental cell line, U-2 OS-WT, and its resistant counterpart, U-2 OS-R. A differential effect between sensitive and resistant cells on the intracellular DOX concentration and distribution was revealed. In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells. Moreover, the cell survival analysis and the electron microscopic observations revealed an enhancement of the cytotoxic effect of DOX induced by the plant extract. In the present study, a panel of monoclonal antibodies (MAbs), recognizing different and specific structural and functional state of Pgp, was used. By flow cytometry and immunofluorescence confocal microscopy, a dose-dependent increase of the reactivity of Pgp with MAb UIC2, which specifically recognizes an epitope of the drug transporter in its functional conformation, was detected in voacamine-treated U-2 OS-R cells. Conversely, the expression of the epitope recognized by MAb MC57 was downregulated while MAb MM4.17 did not change its binding level to treated and untreated MDR cells. These data suggest that the plant extract reacts with Pgp producing conformational changes with consequent epitope modulation. Taken together, our observations seem to demonstrate that voacamine is a substrate for Pgp and, therefore, interferes with the Pgp-mediated drug export, acting as a competitive antagonist of cytotoxic agents.

Voacamine, a bisindolic alkaloid from Peschiera fuchsiaefolia, enhances the cytotoxic effect of doxorubicin on multidrug-resistant tumor cells.

Multidrug-resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the chemotherapy. In order to overcome MDR, substances, such as verapamil and cyclosporin A (CsA), were employed. As these P-gp modulating agents did not seem promising in clinical practice, new compounds with a low degree of undesirable side effects, were introduced. In this study, bisindolic alkaloid voacamine was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on drug resistant cells. Two different pairs of tumor cell lines were analyzed: the parental lymphoblastoid cell line CEM-WT and its MDR derivative CEM-R, the parental osteosarcoma cell line U-2 OS-WT and its resistant counterpart U-2 OS-R. These cell lines were characterized for their morphological features by scanning electron microscopy (SEM) and for the expression of the main drug transporters by flow cytometric analysis. The effects of voacamine on the cell survival and on both accumulation and efflux of DOX were then investigated. The intracellular distribution of DOX, given alone or in association with CsA or voacamine, was observed by laser scanning confocal microscopy. A differential effect of voacamine between sensitive and resistant cells on the intracellular DOX concentration and distribution was shown. In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells. The results of cell survival experiments revealed an enhancement of the cytotoxic effect of DOX induced by voacamine, confirmed by evident morphological changes observed by SEM. These findings suggest promising applications of this natural substance against MDR tumors.

Subcellular detection and localization of the drug transporter P-glycoprotein in cultured tumor cells.

In vitro studies on the cellular location of P-glycoprotein (Pgp) are reported with the aim to clarify the relationship between its intracellular expression and the multidrug resistance (MDR) level of tumor cells. Pgp was found abnormally expressed on the plasma membrane of tumor cells with "classical" MDR phenotype. However, Pgp was also often detected on the nuclear envelope and on the membrane of cytoplasmic organelles. The hypothesis that this drug pump maintains a transport function when located in these compartments, is still under debating. Our results, together with those obtained by other researchers, demonstrate that cytoplasmic Pgp regulates the intracellular traffic of drugs so that they are no more able to reach their cellular targets. In particular, we revealed that in MDR breast cancer cells (MCF-7) a significant level of Pgp was expressed in the Golgi apparatus. A similar result was found in human melanoma cell lines, which never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. A strict relationship between intracellular Pgp and intrinsic resistance was demonstrated in a human colon carcinoma (LoVo) clone, which did not express the drug transporter on the plasma membrane. Finally, a structural and functional association between Pgp and ERM proteins has been discovered in drug-resistant human T- lymphobastoid cells (CEM-VBL 100). Our findings strongly suggest a pivotal role of the intracytoplasmic Pgp in the transport of drugs into cytoplasmic vesicles, thus actively contributing to their sequestration and transport outwards the cells. Thus, intracellular Pgp seems to represent a complementary protective mechanism of tumor cells against cytotoxic agents.

Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells.

Drug resistance, one of the major obstacle in the successful anticancer therapy, can be observed at the outset of therapy (intrinsic resistance) or after exposure to the antitumor agent (acquired resistance). To gain a better insight into the mechanisms of intrinsic resistance we have analyzed two human cell types derived from untreated tumors: MCF-7 breast cancer and A549 non small cell lung cancer (NSCLC). We have examined: the cytotoxic effect induced by doxorubicin (DOX); the time course of drug accumulation by flow cytometry and intracellular drug distribution by confocal microscopy; the expression and distribution of proteins related to anthracycline resistance, such as P-gp (P-glycoprotein), MRP1 (multidrug resistance-associated protein) and LRP (lung resistance-related protein). The cytotoxicity assays showed that A549 cells were less sensitive than MCF-7 cells to the DOX treatment in agreement with the different DOX uptake. Moreover, while in A549 cells DOX was mostly located in well defined intracytoplasmic vesicles, in MCF-7 cells it was mainly revealed inside the nuclei. The analysis of P-gp and MRP expression did not show significant differences between the two cell lines while a high expression of LRP was detected at the nuclear envelope and cytoplasmic levels in A549 cells. These findings suggest that the lower sensitivity to DOX treatment showed by lung carcinoma cells could be ascribed to drug sequestration by LRP inside the cytoplasmic compartments.

What is the relationship between P-glycoprotein and adhesion molecule expression in melanoma cells?

A number of studies have reported that increased P-glycoprotein expression in drug-resistant tumour cells may be associated with decreased expression of a family of surface glycoproteins. However, despite its potential biological and clinical relevance, this phenomenon has not been extensively studied. In this study the phenotypic alterations that are associated with the acquisition of the multidrug-resistant phenotype in tumour cells, together with drug transporter overexpression, were investigated in human melanoma cells. The expression of cell adhesion molecules was analysed in a panel of multidrug-resistant melanoma cell lines (M14Dx) showing different degrees of resistance to doxorubicin and different levels of the expression of the drug transporter P-glycoprotein. In particular, expression of intercellular adhesion molecule-1 (ICAM-1), CD44, very late activation antigen (VLA)-5 and VLA-2 was determined by flow cytometry in the different resistant cell lines. A progressive downregulation of all the adhesion molecules examined was revealed in M14Dx cells, in parallel with an increasing level of expression of the drug transporter P-glycoprotein. The results obtained raise the question of the role of P-glycoprotein in the invasive and metastatic behaviour of tumour cells.

Intracellular P-glycoprotein in multidrug resistant tumor cells.

The overexpression of the drug-efflux molecular pump P-glycoprotein (P-gp) may confer to tumor cells the multidrug resistant (MDR) phenotype, which is one of the causes of cancer chemotherapy failure. By investigating several in vitro models of human tumor cells, we observed that P-gp, in addition to its localization on the plasma membrane, can also be found intracellularly. In particular, by using immunocytochemical and cytofluorimetric methods, we revealed that in MDR breast cancer cells (MCF-7) a significant level of P-gp was expressed in the Golgi apparatus, which is the major site of accumulation of the antitumoral compound doxorubicin. Moreover, we demonstrated the intracellular location of P-gp in three stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express the transporter molecule on the plasma membrane. Double immunofluorescence labelling and immunoelectron microscopy revealed, also in this tumor cell type, the location of P-gp in the Golgi apparatus where it seems to play a pivotal role in intracellular drug transport. Finally, we analyzed the expression, localization and function of drug transport proteins in human colon carcinoma lines (LoVo) exhibiting different degrees of intrinsic or drug-induced resistance. We found that only MDR LoVo cells expressed P-gp on the plasma membrane while both low-level drug resistant clonal LoVo cells and MDR LoVo cells appeared to be positive for intracellular P-gp. Our findings suggest a functional role of the intracytoplasmic P-gp in the transport and sequestration of drugs. This represents a complementary protective mechanism of tumor cells against cytotoxic agents.

1H NMR-visible mobile lipid domains correlate with cytoplasmic lipid bodies in apoptotic T-lymphoblastoid cells.

The presence of nuclear magnetic resonance (NMR)-visible mobile lipid (ML) domains in apoptotic lymphoblasts suggests alterations in neutral lipid metabolism and compartmentation during programmed cell death. The detection of similar ML signals in activated lymphocytes raises questions about common mechanisms of ML formation during apoptosis and upon lymphoblast stimulation. Structure and subcellular localization of ML domains were therefore investigated by NMR, fluorescence and electron microscopy in Jurkat T-lymphoblasts either induced to apoptosis (by anthracyclines or dexamethasone or by serum deprivation) or activated by phorbol myristate acetate (PMA) plus ionomycin. ML contents in drug-treated cells correlated linearly with apoptosis, irrespective of the specific inducer and cell cycle arrest phase (r = 0.993, P < 0.001). Similar ML levels were measured in drug-induced apoptotic cells (A approximately 30-40%) and in non-apoptotic PMA/ionomycin-treated lymphoblasts (72 h). Lower ML contents were instead formed in serum-deprived apoptotic cells, with respect to controls. Increases in ML signals were associated, in either apoptotic or activated cells, with the accumulation of cytoplasmic, osmophilic lipid bodies (diameter < or = 1.0 microm), surrounded by own membrane, possessing intramembrane particles. The results support the hypothesis that ML are formed in the cytoplasm of drug-induced apoptotic cells during an early, 'biochemically active' phase of programmed cell death.

The relationship between 1H-NMR mobile lipid intensity and cholesterol in two human tumor multidrug resistant cell lines (MCF-7 and LoVo).

The high resolution proton nuclear magnetic resonance (1H-NMR) spectra of two different cell lines exhibiting multidrug resistance (MDR) as demonstrated by the expression of the well-known energy-driven, membrane-bound 170 kDa P-glycoprotein pump known as Pgp were investigated. In particular, the mobile lipid (ML) profile, and the growth and biochemical characteristics of MCF-7 (human mammary carcinoma) and LoVo (human colon adenocarcinoma) sensitive and resistant tumor cells were compared. The results indicate that both MCF-7 and LoVo resistant cells have a higher ML intensity than their respective sensitive counterparts. However, since sensitive and resistant cells of each pair grow in the same manner, variations in growth characteristics do not appear to be the cause of the ML changes as has been suggested by other authors in non-resistant tumor cells. In order to investigate further the origin of the ML changes, lipid analyses were conducted in sensitive and resistant cell types. The results of these experiments show that resistant cells of both cell types have a greater amount of esterified cholesterol and saturated cholesteryl ester and triglyceride fatty acid than their sensitive counterparts. From a thorough analysis of the data obtained in this paper utilizing numerous techniques including biological, biophysical and biochemical ones, it is hypothesized that cholesterol and triglyceride play a pivotal role in inducing changes in NMR ML signals. The importance of these lipid variations in MDR is discussed in view of the controversy regarding the origin of ML signals and the paramount role played by the Pgp pump in resistance.

Intracellular P-glycoprotein expression is associated with the intrinsic multidrug resistance phenotype in human colon adenocarcinoma cells.

The 2 clones, LoVo 5 and LoVo 7, derived from untreated LoVo WT human colon adenocarcinoma cells and exhibiting different sensitivity to doxorubicin (DOX), were compared in order to identify possible determinants of intrinsic drug resistance. A multidrug resistant variant cell line, selected from LoVo WT cells by continuous exposure to DOX (LoVo DX), was also included in the study. Analysis of the expression and organization of cytoskeletal elements by flow cytometry and fluorescence microscopy evidenced a positive correlation between vimentin expression and DOX resistance in LoVo 7 and LoVo DX cells, whereas differences in actin, tubulin or cytokeratin did not seem to relate to drug response. The expression and localization of different drug transporters commonly implicated in drug resistance, i.e., the MDR1 gene product P-glycoprotein (P-gp), the multidrug resistance-related protein MRP and the lung resistance-related protein LRP were also investigated by means of flow cytometry and fluorescence microscopy, following labeling with specific monoclonal antibodies. Surface expression of P-gp was only detected in LoVo DX cells, which also exhibited increased MRP and LRP protein levels. However, significant amounts of P-gp were found at intracellular sites in the intrinsically resistant LoVo 7 clone. Modulation of P-gp function by cyclosporin A was found to alter DOX accumulation and efflux in LoVo 7 cells, indicating that intracellular P-gp plays a functional role in drug trafficking and suggesting possible implications in determining the intrinsic resistance displayed by this clone.

Detection of P-glycoprotein in the nuclear envelope of multidrug resistant cells.

P-glycoprotein is a plasma membrane efflux pump which is responsible for multidrug resistance of many cancer cell lines. A number of studies have demonstrated the presence of P-glycoprotein molecules, besides on the plasma membrane, also in intracellular sites, such as the Golgi apparatus and the nucleus. In this study, the presence and function of P-glycoprotein in the nuclear membranes of human breast cancer cells (MCF-7 WT) and their multidrug resistant variants (MCF-7 DX) were investigated. Electron and confocal microscopy immunolabelling experiments demonstrated the presence of P-glycoprotein molecules in the nuclear membranes of MCF-7 DX cells. Moreover, the labelling pattern was strongly dependent on pH values of the incubation buffer. At physiological pH (7.2), a strong labelling was detected in the cytoplasm and the nuclear matrix in both sensitive and resistant MCF-7 cells. By raising the pH to 8.0, the P-glycoprotein molecules were easily detected in the cytoplasm (transport vesicles and Golgi apparatus), plasma and nuclear membranes exclusively in MCF-7 DX cells. Furthermore, drug uptake and efflux studies, performed by flow cytometry on isolated nuclei in the presence of the P-glycoprotein inhibitor cyclosporin A, suggested the presence of a functional P-glycoprotein in the nuclear membrane, but not in the nuclear matrix, of drug resistant cells. Therefore, P-glycoprotein in the nuclear envelope seems to represent a further defense mechanism developed by resistant cells against antineoplastic agents.

Biophysical and structural characterization of 1H-NMR-detectable mobile lipid domains in NIH-3T3 fibroblasts.

Nature and subcellular localization of 1H-NMR-detectable mobile lipid domains (ML) were investigated by NMR, Nile red fluorescence and electron microscopy, in NIH-3T3 fibroblasts and their H-ras transformants (3T3ras) transfected with a high number of oncogene copies. Substantial ML levels (ratio of (CH2)n/CH3 peak areas R=1. 56+/-0.33) were associated in untransformed fibroblasts with both (a) intramembrane amorphous lipid vesicles, about 60 nm in diameter, distinct from caveolae; and (b) cytoplasmic, osmiophilic lipid bodies surrounded by own membrane, endowed of intramembrane particles. 2D NMR maps demonstrated that ML comprised both mono- and polyunsaturated fatty chains. Lower ML signals were detected in 3T3ras (R=0.76+/-0.37), under various conditions of cell growth. Very few (if any) lipid bodies and vesicles were detected in the cytoplasmic or membrane compartments of 3T3ras cells with R<0.4, while only intramembrane lipid vesicles were associated with moderate R values. Involvement of phosphatidylcholine hydrolysis in ML generation was demonstrated by selective inhibition of endogenous phospholipase C (PC-plc) or by exposure to bacterial PC-plc. This study indicates that: (1) both cytoplasmic lipid bodies and membrane vesicles (possibly in mutual dynamic exchange) may contribute (although to a different extent) to ML signals; and (2) high levels of ras-transfection either inhibit ML formation or facilitate their extrusion from the cell.

Detection of P-glycoprotein in the Golgi apparatus of drug-untreated human melanoma cells.

The intracellular location of the MDR1 gene product, known as P-glycoprotein (P-gp), has been detected by flow cytometry in 3 stabilized human melanoma cell lines which had never undergone cytotoxic drug treatment and did not express P-gp on the plasma membrane. In addition, MDR1 mRNA expression was revealed by RT-PCR in the same cell lines. Immunofluorescence microscopy, performed by using the same 2 monoclonal antibodies (MM4.17 and MRK-16) as employed in the flow-cytometric analysis, revealed the presence of P-gp intracytoplasmically, in a well-defined perinuclear region. Double immunofluorescence labelling and immunoelectron microscopy strongly suggested the location of the transporter molecule in the Golgi apparatus. The same observations have been obtained on a primary culture from a metastasis of human melanoma. Analysis of the expression of another membrane transport protein, the multidrug-resistance-related protein (MRP1), showed that it was present in the cytoplasm of all the melanoma cell lines examined. MRP1 also showed Golgi-like localization. The study by laser scanning confocal microscopy on the intracellular localization of the anti-tumoral agent doxorubicin (DOX) during the drug-uptake and -efflux phases, indicated the Golgi apparatus as a preferential accumulation site for the anthracyclinic antibiotic. P-gp function modulators (verapamil and cyclosporin A) were able to modify DOX intracytoplasmic distribution and to increase drug intracellular concentration and cytotoxic effect in melanoma cells. On the contrary, MRP1 modulators (probenecid and genistein) did not significantly influence either DOX efflux and distribution or the sensitivity of melanoma cells to the cytotoxic drug.

Intracellular distribution of anthracyclines in drug resistant cells.

The unresponsiveness of multidrug resistant tumor cells to antineoplastic chemotherapy is often associated with reduced cellular drug accumulation accomplished by overexpressed transport molecules. Moreover, intracellular drug distribution in resistant cells appears to be remarkably different when compared to their wild type counterparts. In the present paper, we report observations on the intracellular accumulation and distribution of doxorubicin, an antitumoral agent widely employed in chemotherapy, in sensitive and resistant cultured tumor cells. The inherent fluorescence of doxorubicin allowed us to follow its fate in living cells by laser scanning confocal microscopy. This study included flow cytometric analysis of drug uptake and efflux and analysis of the presence of the well known drug transporter P-glycoprotein. Morphological, immunocytochemical and functional data evidentiated the Golgi apparatus as the preferential intracytoplasmic site of drug accumulation in resistant cells, capable of sequestering doxorubicin away from the nuclear target. Moreover, P-glycoprotein has been found located in the Golgi apparatus in drug induced resistant cells and in intrinsic resistant cells, such as melanoma cells. Thus, this organelle seems to play a pivotal role in the intracellular distribution of doxorubicin.

Lower levels of 1H MRS-visible mobile lipids in H-ras transformed tumorigenic fibroblasts with respect to their untransformed parental cells.

High resolution 1H MRS studies report increased mobile neutral lipid (MNL) signals in transformed and malignant as well as in some in vitro cultured embryonic cells. Nature, subcellular localization and biological function of MNL are still under debate. This work was aimed at assessing alterations induced in MNL signals of NIH-3T3 mouse embryo fibroblasts by transformation with human HJ-ras oncogene. Lower MRS-visible MNL levels were unexpectedly detected in ras-transformed, in vivo tumorigenic fibroblasts, with respect to their untransformed and non-tumorigenic parental cells. MRS, gas chromatography and chemical analysis on cells and their lipid extracts indicated that these spectral differences could hardly be attributed to different triacylglycerol, free fatty acids and total cholesterol levels or to changes in the fatty acyl degree of unsaturation and average chain length. Additional, possibly more relevant mechanisms of regulation of MNL mobility may implicate the extensive morphogenetic changes and reorganization of cytoskeleton components (notably actin) associated with ras-transformation.