Gloria M. Martinez

pH and drug resistance. I. Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines.

A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells. Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump. However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities. Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance. In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells. Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX). The resting steady-state pHi was similar in the three cell lines. In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity. These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines. The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated. In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did. Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors. Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane. Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells. Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not. Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover. The potential impact of this behavior on drug resistance is examined in a companion manuscript.

DISTINCT REGULATION OF PHIN AND CA2+IN IN HUMAN MELANOMA CELLS WITH DIFFERENT METASTATIC POTENTIAL

We investigated whether alterations in the mechanisms involved in intracellular pH (pHin) and intracellular calcium ([Ca2+]in) homeostasis are associated with the metastatic potential of poorly (A375P) and highly (C8161) metastatic human melanoma cells. We monitored pHin and [Ca2+]in simultaneously, using the fluorescence of SNARF‐1 and Fura‐2, respectively. Our results indicated that steady‐state pHin and [Ca2+]in between these cell types were not significantly different. Treatment of cells with NH4Cl resulted in larger pHin increases in highly than in poorly metastatic cells, suggesting that C8161 cells have a lower H+ buffering capacity than A375P. NH4Cl treatment also increased [Ca2+]in only in C8161 cells. To determine if the changes in [Ca2+]in triggered by NH4Cl treatment were due to alterations in either H+‐ or Ca2+‐buffering capacity, cells were treated with the Ca2+‐ionophore 4Br‐A23187, to alter [Ca2+]in. The magnitude of the ionophore‐induced [Ca2+]in increase was slightly greater in C8161 cells than in A375P. Moreover, A375P cells recover from the ionophore‐induced [Ca2+]in load, whereas C8161 cells did not, suggesting that A375P may exhibit distinct [Ca2+]in regulatory mechanisms than C8161 cells, to recover from Ca2+ loads. Removal of extracellular Ca2+ ([Ca2+]ex) decreased [Ca2+]in in both cell types at the same extent. Ionophore treatment in the absence of [Ca2+]ex transiently increased [Ca2+]in in C8161, but not in A375P cells. Endoplasmic reticulum (ER) Ca2+‐ATPase inhibitors such as cyclopiazonic acid (CPA) and thapsigargin (TG) increased steady‐state [Ca2+]in only in C8161 cells. Together, these data suggest that the contribution of intracellular Ca2+ stores for [Ca2+]in homeostasis is greater in highly than in poorly metastatic cells. Bafilomycin treatment, to inhibit V‐type H+‐ATPases, corroborated our previous results that V‐H+‐ATPases are functionally expressed at the plasma membranes of highly metastatic, but not in poorly metastatic cells in and [Ca2+]in regulatory mechanisms are present in poorly and highly metastatic human melanoma cells. J. Cell. Physiol. 176:196–205, 1998.

Plasmalemmal vacuolar H+-ATPase is decreased in microvascular endothelial cells from a diabetic model.

Angiogenesis requires invasion of extracellular matrix (ECM) proteins by endothelial cells and occurs in hypoxic and acidic environments that are not conducive for cell growth and survival. We hypothesize that angiogenic cells must exhibit a unique system to regulate their cytosolic pH in order to cope with these harsh conditions. The plasmalemmal vacuolar type H+‐ATPase (pmV‐ATPase) is used by cells exhibiting an invasive phenotype. Because angiogenesis is impaired in diabetes, we hypothesized that pmV‐ATPase is decreased in microvascular endothelial cells from diabetic rats. The in vitro angiogenesis assays demonstrated that endothelial cells were unable to form capillary‐like structures in diabetes. The proton fluxes were slower in cells from diabetic than normal model, regardless of the presence or absence of Na+ and HCO  3− and were suppressed by V‐H+‐ATPase inhibitors. Immunocytochemical data revealed that pmV‐ATPases were inconspicuous at the plasma membrane of cells from diabetic whereas in normal cells were prominent. The pmV‐ATPase activity was lower in cells from diabetic than normal models. Inhibition of V‐H+‐ATPase suppresses invasion/migration of normal cells, but have minor effects in cells from diabetic models. These novel observations suggest that the angiogenic abnormalities in diabetes involve a decrease in pmV‐ATPase in microvascular endothelial cells.

Mag-Fura-2 (Furaptra) Exhibits both Low (µM) and High (nM) Affinity for Ca2+

Based on studies using high-affinity Ca²⁺ probes (dissociation constant (Kd) = 0.15–0.3 µM), steady-state [Ca²⁺]ⁱⁿ is believed to be in the nanomolar range in most cells. However, probes with lower affinity indicate that [Ca²⁺]ⁱⁿ may increase to micromolar levels during activation of specific cell functions, e.g., contraction. These conclusions rely on accurate knowledge of the Kd of the dyes for Ca²⁺. Mag-Fura-2 (also known as Furaptra) is a low-affinity Ca²⁺ indicator (Kd ca. 50 µM) which has been used for such studies. In the present work, Mag-Fura-2 is shown to respond to changes in cytosolic Ca²⁺ in the submicromolar range. In vitro, and in situ titration of Mag-Fura-2 in A7r5 cells, demonstrate that Mag-Fura-2 exhibits both high- and low-affinity for Ca²⁺. Moreover, pH affects both high and low affinity Ca²⁺ binding site. Since Mag-Fura-2 has been used to study Ca²⁺ within specific subcellular compartments, the present observations indicate that knowledge of factors such as ambient pH of these compartments is required to accurately interpret Ca²⁺ responses. Furthermore, the sensitivity of Mag-Fura-2 at submicromolar levels must be considered for accurate determination of Ca²⁺ in specific compartments believed to exhibit high micromolar levels of Ca²⁺.

Use of enzyme-developed immunoblots scanned by densitometry to compare lot-to-lot variability of Russian-thistle extracts.

Two different lots of Russian-thistle extracts from five commercial sources were evaluated by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblots of Immobilon transfers from these separations. The immunoblots were developed by an enzyme-linked method and scanned with a densitometer. The two lots of a standardized extracts produced scans that suggested uniformity in allergenic components. Several of the nonstandarized extract lots also appeared quite comparable. With further refinement, immunoblotting with densitometric analysis appears to offer an attractive way to standardize allergen extracts.

Cytosolic pH gradients in cultured neuronal cell lines studied by laser scanning confocal microscopy, real-time confocal microscopy, and spectral imaging microscopy

Changes in intracellular pH are important for the regulation of many physiological processes including: cell growth and differentiation, exocytosis, synaptic transmission, cell motility and invasion, to name a few. In pathological states such as cancer and diabetes, pH regulation is known to be altered. Nevertheless the physiological and pathological significance of this ion, there are still many gaps in our knowledge. The advent of fluorescent pH probes to monitor this ion, has substantially accelerated its study. New advances in the methods of detection of this ion by fluorescence-based approaches have also helped us to understand more about the regulation of cytosolic pH. This study evaluates the usefulness of real time confocal imaging microscopy, laser scanning confocal microscopy, and spectral imaging microscopy to the study of pH. These approaches exhibit unsurpassed temporal, spatial, and spectral resolution and are complementary. We employed cell lines derived from the brain exhibiting soma and dendrites. The existence of cell polarity suggests that the different protein composition/micro environment in discrete subcellular domains may affect the properties of fluorescent ion indicators. We performed in situ calibration of pH probes in discrete cellular regions of the neuronal cell lines to eliminate any bias in data interpretation because of differences in cell thickness/micro environment. We show that there are distinct in situ calibration parameters in different cellular domains. These indicate that in situ titrations in discrete cellular domains are needed to assign pH values. We concluded that there are distinct pH micro domains in discrete cellular regions of neuronal cell lines.

Proton pumps, angiogenesis, and metastatic breast cancer

We have previously shown the relationship between metastatic potential and plasmalemmal V-H+-ATPase (pmV-ATPase) expression in tumor cells. This led us to hypothesize that pmV-ATPase activity is involved in invasion. Angiogenesis involves invasion of adjacent tissues by microvascular endothelial cells, thus we hypothesized that pmV-ATPases contribute to pHin regulation and invasion in microvascular endothelial cells.

Fluorescent pH probes, fluorescent proteins, and intrinsic cellular fluorochromes are tools to study cytosolic pH (pHcyt) in mammalian cells.

Our understanding of intracellular pH homeostatis in eukaryotic systems has been enhanced since the introduction of carboxyfluorescein diacetate as a useful pH probe more than 20 years ago. BCECF, a derivative of this earlier fluoroprobe has dominated the field. In the past 10 years, SNARF-1 has emerged as an alternative pH probe. Recently, a novel derivative of BCECF, BCPCF has been developed. Green Fluorescent Proteins (GFPs) have also been used recently to monitor pH in a non invasive manner in several cell types. Here, we report that human mammary epithelial cells can be transfected with the gene encoding for cyan (CFP), green (GFP), and yellow (YFP), to study cytosolic pH. The novel red fluorescent protein (DsRed) is not sensitive to pH. Multidrug resistance (MDR) has been associated with altered cytosolic pH homeostasis. We show that experimental maneuvers that decrease pHin enhance the efficacy of chemotherapeutic drugs. We also show that short pulses of UV-B light elicited acidosis in cells, as evaluated by ratio ion cell imaging, and confocal/spectral imaging microscopy. During the course of these experiments we noticed that cells exhibit intrinsic fluorochromes that can be used to monitor pH in living cells.