The mitochondrial probe rhodamine 123 inhibits in isolated hepatocytes the degradation of short-lived proteins

The fluorescent dye rhodamine 123 (R123) decreases the intracellular ATP levels and also inhibits the degradation of short-lived proteins in isolated hepatocytes. This inhibition affects lysosomal and, to some extent, non-lysosomal mechanisms. The degradation of short-lived proteins decreases more when ATP levels are less than 40% of those in control cells, in contrast to the reported linear correlation between ATP levels and degradation of long-lived proteins. R123 provides a powerful probe for clarifying the proteolytic mechanisms involved in degradation of short-lived proteins and the ATP requirements in protein degradation. Indeed, as illustrated, the results suggest different mechanisms for the degradation of short- and long-lived proteins. Moreover, they provide a warning for the clinical use of this reagent.     

Induction of osteoprogenitor cell differentiation in rat marrow stroma increases mitochondrial retention of rhodamine 123 in stromal cells

Bone marrow stromal cells contain colony forming cells with the potential to differentiate into osteoprogenitor (OPC) cells. OPC-stimulation medium, containing dexamethasone, ascorbate, and β-glycerophosphate is widely used to recruit OPCs in culture. Cultures were incubated 24 h with rhodamine 123 (Rho), on different days, to examine the effect of the OPC-stimulation medium on the mitochondrial membrane potential of stromal cells. Cultures grown in both ordinary medium (DMEM with 15% FCS) and OPC-stimulation medium showed 2 Rho retention peaks on days 3–4 and 10–11. Between days 5 and 10 there was a drop in Rho retention/cell. OPC-stimulation medium increased Rho retention by at least twice the amount relative to ordinary medium, and has quadrupled it on day 7. Incubation with Rho concentrations above 5.0 μg/ml inhibited the portion of increased Rho retention which was contributed by the OPC-stimulation medium. Prolonged exposure to 0.1, 1.0, and 10.0 μg/ml Rho for 12 days only slightly increased day 12 ALP activity/cell, had no effect on day-21 mineralization and only the high dose, 10.0 μg/ml, doubled stromal cell proliferation. Under 24 h incubation Rho concentrations of 1.0 μg/ml and below can serve as a marker for mitochondrial membrane potential in differentiating stromal cells. The results indicate that under both culture conditions stromal cell mitochondria undergo cycles of high and low membrane potential states and that the OPC-stimulation medium constantly maintains an elevated membrane potential relative to ordinary medium.     

Isolation and enrichment of murine spermatogonial stem cells using rhodamine 123 mitochondrial dye

Stem cells possess enormous therapeutic potential in tissue replacement. To study stem cells further, they must be isolated. Techniques are available for enrichment and study of hematopoietic stems cells, but thus far, techniques for purification of spermatogonial stem cells have not been described. Enrichment techniques for hematopoietic stem cells include the use of fluorescence-activated cell sorter analysis with Hoechst 33342 and rhodamine 123 (Rho) dyes. Use of Hoechst dye to isolate spermatogonial stem cells has been unsuccessful in our laboratory, and our results have conflicted with those from other laboratories. Taking advantage of the differential staining of the Rho dye, we report a novel method to enrich murine spermatogonial stem cells. Testicular cells are harvested from cryptorchid ROSA26 male mice. Populations of these cells are then stained with the Hoechst and Rho dyes, allowing them to be sorted by flow cytometry into a side population (SP) of Hoechst low-intensity cells and populations of low (Rho(low)) or high (Rho(hi)) fluorescent intensity. Sterile recipients, W/W(v) mice, with an intrinsic germ cell deficiency were transplanted with the Hoechst SP cells, Rho(low), Rho(hi), and nonsorted donor cells. No spermatogonial stem cell colonies were derived from the Hoechst SP cells. The number of spermatogonial stem cell colonies from transplanted Rho(low) cells showed a 17- and 20-fold enrichment over those of Rho(hi) and nonsorted cells, respectively.     

Use of nonyl acridine orange and rhodamine 123 to follow biosynthesis and functional assembly of mitochondrial membrane during L1210 cell cycle.

Specific mitochondrial incorporation of 10 N-nonyl acridine orange (NAO) is demonstrated by subcellular fractionation of rat hepatocytes. Moreover, comparative studies with NAO and rhodamine 123 (Rh 123) prove that acridine orange-derivative uptake is independent of transmembrane mitochondrial potential, a property allowing its utilization for the assessment of mitochondrial membrane mass modifications under various physiological states. Using NAO and Rh 123, we have respectively followed the biosynthesis of mitochondrial membrane and its assembly under a functional state during the L1210 cell cycle. Their evolution occurs in two stages according to a well-defined sequential order. Mitochondrial biogenesis, as revealed by NAO incorporation, occurs essentially in the G1 phase (probably mitochondrion enlargement) but also starts in late S phase (probably mitochondrion division). The increased amount of functional mitochondrial membrane, monitored by Rh 123 uptake, is emphasized in late G1 (prerequisite to DNA synthesis) and during G2M phases (prerequisite to mitosis). This alternative succession of phases displays the existence of a time-lag between the biosynthesis of mitochondrial membrane and its functional organization. Such an analysis confirms the potential of the NAO probe to evaluate mitochondrial membrane mass changes in various biological fields.     

Interference of calmidazolium with measurement of mitochondrial membrane potential using the tetraphenylphosphonium electrode or the fluorescent probe rhodamine 123

Calmidazolium (CMZ) is a positively charged, hydrophobic compound used as a calmodulin antagonist. It may cause unspecific effects in mitochondria, e.g., a decrease in membrane potential (deltapsi), swelling, and uncoupling. Several groups have advised against use of CMZ in studying signal transduction in mitochondria. We report here that it interferes with measurement of deltapsi in rat liver mitochondria (RLM) when using the tetraphenyl phosphonium (TPP+) electrode. We also found that CMZ reduces the signal, indicating an apparent drop in deltapsi. CMZ itself gave a signal with the TPP+ electrode in the absence of RLM. At high concentrations, > 10 microM, it also reduced the fluorescence quenching of the probe rhodamine 123. This may be due to an interference with mitochondrial uptake and binding of this positively charged probe or to an uncoupling effect. It is concluded that CMZ and similar positively charged calmodulin antagonists such as trifluoperazine may be used in mitochondria if these interferences are controlled and calibration is carried out under the experimental conditions used.     

Terminal deoxynucleotidyl transferase in AKR leukemic cells and lack of relation of enzyme activity to cell cycle phase.

Cholate and taurocholate uptakes were studied in presence of albumin using isolated rat hepatocytes. Albumin decreased nonspecific binding of both bile acids and inhibited cholate uptake noncompetitively and taurocholate uptake competitively. Although different bile acids except dehydrocholate inhibited both cholate and taurocholate uptake, their relative inhibitory potency was not the same for both bile acids. Uptake of both bile acids was characterized by a saturable as well as an unsaturable process both in presence and in absence of albumin. The results suggest that both bile acids may be transported by more than one carrier and taurocholate is transported more efficiently than cholate by hepatocytes.     

Use of rhodamine 123 to investigate alterations in mitochondrial activity in isolated mouse liver mitochondria

The fluorescent dye Rhodamine-123, which selectively stains mitochondria depending on the mitochondrial membrane potential, was used with flow cytometry to evaluate alterations in activity of mitochondria isolated from mouse liver. Under in vitro conditions, with succinate and ADP present in the buffer, mitochondrial activity was affected by a variety of metabolic inhibitors that modify membrane potential. These results demonstrate clearly that flow cytometric techniques using Rhodamine-123 can be employed to study activity in isolated mitochondria.     

The use of retinoic acid to probe the relation between hyperproliferation-associated keratins and cell proliferation in normal and malignant epidermal cells

When cells from normal human epidermis and from the human squamous cell carcinoma line SCC-13 were seeded on floating rafts of collagen and fibroblasts, they stratified and underwent terminal differentiation. Although the program of differentiation in SCC-13 cells was morphologically abnormal, the cultures resembled normal epidermal raft cultures by expressing the terminal differentiation-specific keratins, K1/K10, and by restricting their proliferative capacity to the basal-like cells of the population. In addition, the differentiating cells of both normal and SCC-13 raft cultures expressed keratins K6 and K16, which are not normally expressed in epidermis, but are synthesized suprabasally during wound-healing and in various epidermal diseases associated with hyperproliferation. While the behavior of normal and SCC-13 rafts was quite similar when they were cultured over normal medium, significant biochemical differences began to emerge when the cultures were exposed to retinoic acid. Most notably, while the SCC-13 cultures still stratified extensively, they showed a marked inhibition of both abnormal (K6/K16) and normal (K1/K10) differentiation-associated keratins, concomitantly with an overall disappearance of differentiated phenotype. Surprisingly, the reduction in K6/K16 in retinoid-treated SCC-13 cultures was not accompanied by a decrease in cell proliferation. Using immunohistochemistry combined with [3H]thymidine labeling, we demonstrate that while the expression of K6 and K16 are often associated with hyperproliferation, these keratins are only produced in the nondividing, differentiating populations of proliferating cultures. Moreover, since their expression can be suppressed without a corresponding decrease in proliferation, the expression of these keratins cannot be essential to the nature of the hyperproliferative epidermal cell.     

Ganglioside composition of normal and leukemic bovine lymphocytes

The ganglioside composition of bovine peripheral lymphocytes was shown to change sharply under lymphoid leukemia. In normal lymph, lymph nodes, spleen and blood lymphocytes the major ganglioside is N-glycolylhematoside, whereas in calf thymus lymphocytes appreciable amounts of more polar components (GM1- and GD1a-like gangliosides) were found. In leukemic lymphocytes isolated from the same tissues the hematoside content is decreased, while the amount of more polar gangliosides is increased. Possible causes of the altered ganglioside pattern in leukemic lymphocytes are discussed.     

Species-specific differences in the toxicity of rhodamine 123 towards cultured mammalian cells

The toxicity of cationic fluorescent dye, rhodamine 123, towards a number of independently established cell lines from three different species, namely human, mouse, and Chinese hamster, has been examined. All of the cell lines from any one species that were examined were found to exhibit similar sensitivities towards rhodamine 123 and no appreciable differences were observed between the normal and transformed cell types. However, in comparison to the cells of human origin, mouse and Chinese hamster cell lines exhibited about 10-fold and 70-fold higher resistance, respectively, and these differences appeared to be species related. In contrast to rhodamine 123, no differences in relative toxicities for these cell lines were observed for the structurally related neutral dye, rhodamine B. Fluorescence studies with rhodamine 123 show that in comparison to mouse and Chinese hamster cells, the more sensitive human cells show much higher uptake/binding of the drug, and a good correlation was seen in these studies between the extent of dye uptake/binding and the relative sensitivities of cell lines to rhodamine 123. These results provide evidence that the observed species-related differences in cellular toxicities are due to differences in the cellular uptake/binding of the dye.     

Observations on prostaglandins in normal and leukemic human lymphocytes

Prostaglandins E (PGE) and F2 alpha (PGF2 alpha) were measured in lymphocytes of normal subjects, children with acute lymphocytic leukemia (ALL), and adults with chronic lymphocytic leukemia (CLL). In ALL lymphocytes PGE increased from a normal value of 25 pgrams to 270 pgrams/10(6) cells, and PGF 2 alpha increased from a normal value of 31 pgrams to 482 pgrams/10(6) cells. In CLL lymphocytes, levels of PGE and PGF2 alpha were normal or low. When normal lymphocytes were stimulated with phytohemagglutinin (PHA), the level of PGE and PGF2 alpha fluctuated, followed by corresponding changes in the level of cyclic nucleotides. In cultured ALL lymphocytes, the level of PGE remained high, while cyclic 3':5'-adenosine monophosphate (c-AMP) level was constantly low, and the initial level of PGF2 alpha fluctuated in relation to similar oscillations of cyclic 3':5'-guanosine monophosphate (c-GMP). These values were lower, although not significantly, when ALL lymphocytes were stimulated with PHA. When CLL lymphocytes were stimulated with PHA, the level of PGE remained low (20 pgrams), as did that of c-AMP. The level of PGF2 alpha, after a brief initial increase (130 pgrams), returned to and remained at a lower level (60 pgrams) while the level of c-GMP was persistently high. These results suggest: (1) prostaglandins may indirectly influence the cell cycle, possibly through modulation of cyclase activity and levels of cyclic nucleotides; and (2) some derangement of this regulatory mechanism may be present in leukemic lymphocytes.     

Observations on prostaglandins in normal and leukemic human lymphocytes

Prostaglandins E (PGE) and F₂ (PGF₂) were measured in lymphocytes of normal subjects, children with acute lymphocytic leukemia (ALL), and adults with chronic lymphocytic leukemia (CLL). In ALL lymphocytes PGE increased from a normal value of 25 pgrams to 270 pgrams/10⁶ cells, and PGF₂ increased from a normal value of 31 pgrams to 482 pgrams/10⁶ cells. In CLL lymphocytes, levels of PGE and PGF₂ were normal or low. When normal lymphocytes were stimulated with phytohemagglutinin (PHA), the level of PGE and PGF₂ fluctuated, followed by corresponding changes in the level of cyclic nucleotides. In cultured ALL lymphocytes, the level of PGE remained high, while cyclic 3′:5′-adenosine monophosphate (c-AMP) level was constantly low, and the initial high level of PGF₂ fluctuated in relation to similar oscillations of cyclic 3′:5′-guanosine monophosphate (c-GMP). These values were lower, although not significantly, when ALL lymphocytes were stimulated with PHA. When CLL lymphocytes were stimulated with PHA, the level of PGE remained low (20 pgrams), as did that of c-AMP. The level of PGF₂, after a brief initial increase (130 pgrams), returned to and remained at a lower level (60 pgrams) while the level of c-GMP was persistently high. These results suggest: (1) prostaglandins may indirectly influence the cell cycle, possibly through modulation of cyclase activity and levels of cyclic nucleotides; and (2) some derangement of this regulatory mechanism may be present in leukemic lymphocytes.     

Inhibition by rhodamine 123 of protein synthesis in mitochondria of normal and cancer tissues

The effect of the mitochondrial dye rhodamine 123 (Rho 123) on protein synthesis (PS) activity was investigated in mitochondria isolated from liver and from both chloroma and erythroleukemia tumors. Incorporation of labelled leucine into mitochondrial protein was used to measure the rate of PS. While PS specific activity was much higher in hematopoietic tumors mitochondria as compared to that of liver, the addition of increased concentration of Rho 123 in all tested organelles resulted in increased inhibition of PS to reach 75-82% with 10 micrograms/ml of the dye. Similar results were obtained with 10 micrograms/ml of chloramphenicol, the specific inhibitor of mitochondrial PS. Moreover, under the conditions of the study, the addition of Rho 123 to mitochondria did not trigger any ATPase activity, thus eliminating any competition for the energy source ATP between PS and ATPase. These results demonstrate that, in addition to its known inhibitory action on oxidative phosphorylation, the mitochondrial dye Rho 123 has a potent inhibitory effect on PS in both liver and hematopoietic tumors mitochondria.     

Cell volume changes in relation to the cell cycle of differentiating erythroleukemic cells

Murine erythroleukemic cells (MELC) were synchronized by sequential exposure to thymidine and hydroxyurea. Upon removal from hydroxyurea, cells cultured with or without agents that induce erythroid differentiation, such as hexamethylene bisacetamide (HMBA) or dimethylsulfoxide (Me₂SO), proceed through S, G2 and mitosis with the same kinetics: S phase averages 5 h and G2 plus mitosis, 2 h. Cells cultured with HMBA and Me₂SO remain in the subsequent G1 for 5–7 h, compared with an average of only 3 h for cells cultured without inducer. Modal cell volume doubles as the cells proceed from G1 to G2. During the inducer-mediated prolonged G1, MELC retain a small cell volume. In cultures of non-synchronous MELC, inducers also increase the G1 fraction, as well as the proportion of small cells. An Me₂SO-resistant MELC variant (DR10), cultured with Me₂SO, shows little prolongation of G1 and little difference in the modal cell volume compared with cells without inducer. However, HMBA, which induces differentiation of DR10 cells, prolongs G1 and increases the proportion of small cells. These studies indicate that early changes in cell volume associated with induction of MELC to differentiate, in large part reflect alterations in the cell cycle. Evidence is presented which suggests that only one round of DNA synthesis in the presence of inducer may be necessary to initiate differentiation.     

Mitochondrial analysis in living cells: the use of rhodamine 123 and flow cytometry

Flow cytometry combines the advantages of microscopy and biochemical analysis in a single highly sensitive technique for a rapid examination of numerous individual living cells. It has become a potent and essential tool in the studies of the physiology of the whole cell and its organelles. Rhodamine 123 is a vital fluorescent dye used in flow cytometry. As it is specifically concentrated in mitochondria because of the transmembrane potential that these organelles maintain in living cells, rhodamine 123 is thus a useful probe for monitoring the abundance and activity of mitochondria. A critical survey of the routine use of rhodamine 123 together with flow cytometry in mitochondrial research is presented.     

Studies on satellite nucleoli in human normal and leukemic lymphocytes

Lymphocytes from normal and leukemic patients, and phytohemagglutinin-stimulated lymphocytes were investigated by means of immunofluorescence procedures and a silver reaction for the demonstration of proteins characteristic of nucleolus organizer regions in interphasic cells to provide basic information on the presence of satellite nucleoli in these cells. The results clearly indicated that satellite nucleoli are present in a limited but constant percentage of peripheral lymphocytes. An increased percentage of lymphocytes with satellite nucleoli was found only in leukemic patients and after silver staining. In contrast, a decreased percentage of satellite nucleoli was found 24 h after stimulation with phytohemagglutinin vitro. In leukemic patients, the discrepancy in the percentage of lymphocytes with satellite nucleoli between immunostained and silver-stained preparations may suggest that the silver reaction demonstrates the presence of an additional argyrophilic protein besides proteins B23 and C23, or altered forms of these proteins, which does not react with the specific antibodies.