RESUMEN
Gap junctions are transmembrane proteins comprised of six connexin subunits that facilitate direct solute transport between adjacent cells through gap junctions. Previous studies from other laboratories have documented a correlation between reduced gap-junction function and malignant transformation. In endometrial cancer, a characteristic finding is a reduction in the number of stromal cells surrounding the malignant epithelial cells. Thus, the focus of this study was to determine the effect of endometrial stromal cells on gap-junction function in normal and malignant endometrial epithelial cells. To perform these studies, we evaluated normal endometrial epithelial cells and human endometrial epithelial cells including FEEC (fetal endometrial epithelial cells immortalized with simian virus 40 large-T antigen), HEC-1A (endometrial carcinoma stage 1A), and RL-95-2 (endometrial carcinoma grade II). Gap-junctional intercellular communication (GJIC) could not be demonstrated for any of the cell lines. Low levels of GJIC were observed for normal epithelial cells and higher levels were found between stromal cells. Increased levels of GJIC were observed between the epithelial cells when they were cocultured with stromal cells. The transformed epithelial cells showed no GJIC when cultured alone or when in coculture with stromal cells. The results suggest that endometrial stromal cells may help to regulate this differentiated function of endometrial epithelial cells and that malignant endometrial epithelial cells are not responsive to these regulatory signals. Mol. Carcinog. 28:70-75, 2000.
Asunto(s)
Neoplasias Endometriales/patología , Endometrio/citología , Uniones Comunicantes/fisiología , Células del Estroma/citología , Medios de Cultivo , Endometrio/patología , Células Epiteliales/citología , Estradiol/fisiología , Femenino , Humanos , Progesterona/fisiología , Células Tumorales CultivadasRESUMEN
The expression of connexin 43 was studied using immunohistochemical and Western blot analyses on cell lines of endometrial epithelial origin. Connexin proteins were examined because decreases in their expression and function have been correlated with carcinogenesis. The cell lines were chosen to represent increasing grades of endometrial cancer progression starting from FEEC (fetal endometrial epithelial cells; transformed with SV40 large T antigen) to HEC-1A (stage 1A endometrial carcinoma) to RL-95-2 (grade 2 endometrial carcinoma). Parallel studies using connexin 43 polyclonal antibodies for both Western blots and immunofluorescence showed that the levels of connexin 43 expression were normal endometrial stromal cells = FEEC > HEC-1A > RL-95-2. Consequently, we applied the immunofluorescence assay to analyze paraffin-embedded uterine sections from hysterectomy specimens of patients with normal endometrium and from patients diagnosed with grade 1, 2, and 3 endometrial cancer. Using five different cases from each category, we found an inverse correlation between connexin 43 expression and tumor grade. Our data indicate that connexin 43 expression may be useful as an adjunctive marker of progression for endometrial carcinoma.
Asunto(s)
Conexina 43/metabolismo , Neoplasias Endometriales/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Western Blotting , Línea Celular , Neoplasias Endometriales/patología , Endometrio/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Microscopía FluorescenteRESUMEN
A reasonably facile and effective procedure is described for the preparation of inside-out plasma membrane vesicles from tumor cells. The method incorporates nitrogen cavitation, optimized with respect to the applied N2 pressure, in the absence of added divalent cations followed by differential centrifugation and discontinuous, sucrose gradient centrifugation. With the three tumor cell types utilized, multidrug-resistant (MEL/VCR-6) and parental (MEL/O) murine erythroleukemia cells and methotrexate-resistant (L1210/R24) L1210 leukemia cells, yields were in the range of 8-12 mg of plasma membrane vesicles/10(10) cells at a purity of 87-94% with average inside-out sidedness among preparations varying from 65 to 93% depending upon the cell type. Inside-out plasma membrane vesicles so derived were capable of sustaining ATP-dependent transport inward of two common antitumor cytotoxic agents, vinblastine and methotrexate. The former was demonstrated with inside-out vesicles from only P-glycoprotein-overexpressing, multidrug-resistant MEL/VCR-6 cells, while the latter was readily demonstrated in inside-out vesicles from all three cell types.
Asunto(s)
Adenosina Trifosfatasas/farmacología , Leucemia L1210/enzimología , Leucemia Eritroblástica Aguda/enzimología , Animales , Membrana Celular/enzimología , Resistencia a Múltiples Medicamentos , Leucemia L1210/tratamiento farmacológico , Leucemia L1210/patología , Leucemia Eritroblástica Aguda/tratamiento farmacológico , Leucemia Eritroblástica Aguda/patología , Liposomas , Ratones , Nitrógeno , Presión , Células Tumorales CultivadasRESUMEN
Our prior studies with inside-out plasma membrane vesicles from L1210 cells (Schlemmer SR and Sirotnak FM, J Biol Chem 267: 14746-14752, 1992) identified an outwardly directed, translocating ATPase as mediating the majority of methotrexate (MTX) efflux in these cells. In the current studies, we examined by competitive inhibition with [3H]MTX as permeant some of the structural features that determine preferences among folate compounds and their analogues as permeants for this ATPase. The results show that folate compounds are preferred over simple quinazolines (5,8-dideaza-pteridines), and IL5-CH3-folateH4, and probably other 5-substituted folates are preferred over folic acid. In the latter regard, the observed equivalence in preference to IL5-CH3-folateH4 of the 4-oxa-pyridopyrimidine, lometrexol (DDATHF), probably relates to its close similarity to folateH4. The results also suggest that the 4-position in the case of folate analogues, but not in the case of the quinazoline analogues, is an important determinant with 4-amino preferred over 4-oxa. They also suggest that the N10 position on the bridge region in both series of compounds, and probably for the pyridopyrimidine lometrexol, is not an important determinant. In contrast to results seen with the simple quinazolines, the 2-CH3 desamino quinazoline ZEN D1694, modified as well by a 2-benzyl to thienyl replacement on the side chain, was highly preferred. The same relative differences seen among some of these analogues as inhibitors of [3H]MTX efflux in inside-out vesicles were documented for their effectiveness as permeants for ATP-dependent efflux in intact L1210 cells.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ácido Fólico/análogos & derivados , Metotrexato/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Animales , Línea Celular , Membrana Celular/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Cinética , Leucemia L1210/metabolismo , RatonesRESUMEN
Studies with inside-out plasma membrane vesicles from multidrug-resistant (MDR 3) murine erythroleukemia (MEL/VCR-6) cells have provided evidence for down-modulation of P-glycoprotein (P-gp) function by Ca(2+)-calmodulin (CLM). These studies showed that CLM in the presence or absence of Ca2+ had no effect on binding of [3H]vinblastine (VBL) by P-gp in inside-out plasma membrane vesicles. However, profound inhibition of ATP-dependent [3H]VBL efflux by these vesicles was demonstrated by the addition of subnanomolar concentrations of CLM (IC50 = 0.15 +/- 0.02 nM). The addition of 1 microM Ca2+ reduced the inhibition of [3H]VBL efflux by CLM, shifting the concentration required for inhibition to the nM range (IC50 = 2.55 +/- 0.35 nM). The inhibition of as 0.01 mM Ca2+, and no inhibition occurred with concentrations greater than 0.2 mM Ca2+. Binding of CLM, itself, to P-gp was demonstrated in two ways. The P-gp content of detergent-solubilized plasma membrane from MEL/VCR-6 cells could be appreciably depleted by treating this material with CLM-Sepharose beads as shown by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting with anti-P-gp antibody (C219) before and after CLM-Sepharose treatment. Also, depletion of P-gp from solution by CLM was less in the presence of 1 mM Ca2+. Blotting of P-gp after SDS-PAGE of plasma membrane from MEL/VCR-6 cells was also obtained using 125I-CLM as a probe. These results strongly suggest that the MDR 3 homolog of P-gp is a CLM-binding protein and that direct interaction of Ca(2+)-CLM with P-gp, while not affecting its binding of [3H]VBL, down-modulates the translocation of this agent in the presence of ATP.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Calcio/farmacología , Calmodulina/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/aislamiento & purificación , Animales , Western Blotting , Membrana Celular/metabolismo , Resistencia a Múltiples Medicamentos , Cinética , Leucemia Eritroblástica Aguda , Ratones , Factores de Tiempo , Células Tumorales Cultivadas , Vinblastina/metabolismoRESUMEN
Active [3H]vinblastine (VBL) transport (efflux) was documented for inside-out plasma membrane vesicles from murine erythroleukemia cells (MEL/VCR-6) resistant to vinca alkaloids and overexpressing MDR 3 P-glycoprotein (P-gp) 80-fold. Uptake of [3H]VBL at 37 degrees C by these inside-out vesicles, but not rightside-out vesicles or inside-out vesicles from wild-type cells, was obtained in the form of a rapid, initial phase (0-1 min) and a slower, later phase (> 1 min). The rapidity of each phase correlated with relative P-gp content among different MEL/VCR cell lines. The initial MDR-specific phase was temperature- and pH-dependent (optimum at pH 7), osmotically insensitive, and did not require ATP. The second MDR-specific phase was temperature-dependent, osmotically sensitive, and strictly dependent upon the presence of ATP (Km = 0.37 +/- 0.04 mM). Although other triphosphate nucleotides were partially effective in replacing ATP, the nonhydrolyzable analogue ATP gamma S (adenosine 5'-O-(thiotriphosphate)) was ineffective. This time course appears to represent tandem binding of [3H]VBL by P-gp and its mediated transport, with the latter process representing the rate-limiting step. In support of this conclusion, both binding and transport were inhibited by verapamil, quinidine, and reserpine, all known to be inhibitors of photoaffinity labeling of P-gp, but only transport was inhibited by C219 anti-P-gp antibody or orthovanadate. Although the rate of transport of [3H]VBL was 7-7.5-fold lower than the rate of binding (Vmax = 104 +/- 15 pmol/min/mg protein, Kon = 1.5 - 2 x 10(5) mol-1 s-1) to P-gp, each phase exhibited saturation kinetics and values for apparent Km and KD for each process were approximately the same (215 +/- 35 and 195 +/- 30 nM). Intravesicular accumulation of [3H]VBL was almost completely eliminated by high concentrations of nonradioactive VBL, suggesting that simple diffusion does not contribute appreciably to total accumulation of [3H]VBL in this vesicle system. This could be at least partially explained by the fact that these inside-out vesicles under the conditions employed did not maintain a P-gp mediated pH gradient. However, ATP-dependent, intravesicular accumulation of osmotically sensitive [3H]VBL occurred against a substantial permeant concentration gradient in both a time- and concentration-dependent manner consistent with an active, saturable process.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Leucemia Eritroblástica Aguda/metabolismo , Vinblastina/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Resistencia a Múltiples Medicamentos , Concentración de Iones de Hidrógeno , Cinética , Leucemia Eritroblástica Aguda/genética , Ratones , Ratones Endogámicos DBA , Ósmosis , Unión Proteica , Protones , Células Tumorales CultivadasRESUMEN
In the present report, studies are described examining the issue of methotrexate (MTX) polyglutamate retentiveness in cultured L1210 cells. Measurements made in intact L1210 cells showed that the rate of egress of [3H]MTX and [3H]MTX+G1 was different depending upon whether [3H]-MTX and its polyglutamates were accumulated intracellularly following biosynthesis during growth (3 hr) in the presence of [3H]MTX or when cells were pulse loaded (5 min) with [3H]MTX or [3H]-MTX+G1 just prior to measurement of egress. In the former case, [3H]MTX egressed with a T1/2 of 15 +/- 2 min, while [3H]MTX+G1 egressed with a T1/2 of 50 +/- 9 min. In pulse loaded cells, both [3H]MTX and [3H]MTX+G1 egressed with a T1/2 of 3.5 +/- 0.5 min. The same rapid egress of [3H]MTX and [3H]MTX+G1 seen following pulse loading was also documented in cells grown for 3 hr in the presence of nonradioactive MTX to normalize conditions with respect to the intracellular accumulation of [3H]MTX polyglutamates seen during exposure of cells to [3H]MTX during growth. In light of these results, MTX, MTX+G1, MTX+G2 and MTX+G4 were examined directly as permeants for the outwardly-directed ATP-dependent pump (Schlemmer SR and Sirotnak FM, J Biol Chem 267: 14746-14752, 1992) mediating most of MTX efflux in intact L1210 cells using inside-out plasma membrane vesicles isolated from these cells. ATP-dependent efflux of [3H]MTX and [3H]MTX+G1 exhibited values for Km of 46-50 microM and values for Vmax of 102-106 pmol/min/mg protein. As competitive inhibitors of [3H]MTX and [3H]MTX+G1 efflux, MTX+G1 and MTX, respectively, exhibited Ki values of 43-47 microM, that is, Km approximately Ki for both permeants. Also, values for Ki of 45-48 microM were obtained with MTX+G2 and MTX+G4 as competitive inhibitors of [3H]MTX efflux. From these results, we conclude that MTX and its polyglutamates are equivalent as copermeants for ATP-dependent efflux through the plasma membrane and that retentiveness of MTX polyglutamates is not determined at this level in these cells.
Asunto(s)
Membrana Celular/metabolismo , Leucemia L1210/metabolismo , Metotrexato/análogos & derivados , Ácido Poliglutámico/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico , Cinética , Metotrexato/metabolismo , Ácido Poliglutámico/metabolismo , Tritio , Células Tumorales CultivadasRESUMEN
Earlier studies from our laboratory (Dembo, M., Sirotnak F. M., and Moccio, D. M. (1984) J. Membr. Biol. 78, 9-17) suggested that methotrexate (MTX) efflux from L1210 cells was mediated predominantly by an ATP-dependent, outwardly directed, mechanism. To examine this process further, we utilized predominantly (74%) inside-out plasma membrane vesicle preparations derived from an L1210 cell variant (L1210/R24) with 15-fold reduced Vmax for [3H]MTX influx. Efflux of [3H]MTX, under nonionic buffer conditions, in these inside-out membrane vesicles was temperature and ATP dependent (apparent Km = 0.40 +/- 0.06 mM), osmotically sensitive, and unaffected by protonophores. The presence of K+, Na+, Cl-, and HCO3- at their physiological concentrations had no effect on [3H]MTX efflux. Other triphosphonucleotides (GTP and CTP), but not a nonhydrolyzable analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), could also stimulate efflux, but to a lesser extent. Also, ATP gamma S and orthovanadate were potent inhibitors of ATP-dependent efflux of [3H]MTX. Other experiments revealed a system with low saturability for [3H]MTX during efflux (apparent Km = 46 +/- 7 microM), but extremely high capacity (106 +/- 15 pmol/min/mg protein), and a pH optimum in the range of 5.5-6. However, appreciable efflux was measured in the physiological range of pH 6.7-6.9. A number of inhibitors or copermeants for ATP-dependent [3H]MTX efflux in intact L1210 cells were inhibitors of ATP-dependent efflux in inside-out plasma membrane vesicles, including, cholate, bromosulfophthalein, verapamil, quinidine, and reserpine. These findings and other results showing that bromosulfophthalein will completely inhibit efflux are consistent with a role for an ATPase in [3H]MTX efflux, and suggest that the process under study is the bromosulfophthalein-sensitive, ATP-dependent route responsible for the majority of [3H]MTX efflux in intact L1210 cells.