声化学激活血卟啉诱导艾氏腹水肿瘤细胞凋亡

本实验采用频率为2．0MHz，声强分别为1．0w／cm^2、1．5w／cm^2、2．0w／cm^2等不同参数，研究超声激活血卟啉对艾氏腹水肿瘤细胞的杀伤作用和诱导肿瘤细胞凋亡现象。通过扫描电镜、透射电镜以及荧光显微镜观察受损后细胞形态结构的变化，主要表现为细胞微绒毛的减少，胞膜结构和通透性的改变，细胞器的受损以及核物质的分解、丢失；同时发现处理后的肿瘤细胞有核物质凝集、趋边排列以及凋亡小体的形成等细胞凋亡特征。研究中首次发现声化学激活血卟啉在对艾氏腹水肿瘤细胞杀伤的同时，也能诱导艾氏腹水肿瘤细胞发生凋亡，提示在声动力疗法中并存着对癌细胞的直接杀伤和通过诱导癌细胞凋亡的两种抗癌途径。     

Nucleosidediphosphate kinase from Ehrlich ascites tumor cells

A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.     

Mannose toxicity in Ehrlich ascites tumor cells

Mannosephosphate isomerase (MPI) showed a higher activity than hexokinase (HKM) in its ability to phosphorylate mannose in the spleen, thymus, brain, liver, striated muscles, kidneys, and testes from BALB/c mice. This led to a HKM/MPI ratio of less than 1 in all the organs and tissues mentioned. In contrast, Ehrlich ascites tumor cells obtained from the peritoneum of BALB/c mice had low MPI activity (half of the HKM activity and, therefore, a ratio of 2). Mannose, which is nontoxic to nontumor cells at a concentration of 0.1 M, induced marked in vitro mortality of the tumor cells. Incubation of Ehrlich ascites tumor cells with mannose resulted in a high accumulation of mannose-6-phosphate and a marked depletion of ATP which did not appear when the cells were incubated with glucose. These facts may explain the selective mortality caused by mannose in the tumor cells studied.     

DNA ligase from mouse Ehrlich ascites tumor cells

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.     

Characteristics of D-leucine uptake by mouse Ehrlich ascites tumor cells.

In a previous paper, we reported that various D-amino acids were taken up several times more effectively than the corresponding L-isomers into several tumors tested in vivo. In order to investigate this further, the in vitro uptake of D-[14C]leucine by Ehrlich ascites tumor cells was investigated in comparison with that of L-[14C]leucine. The distribution ratio, the effects of amino acids and pH, and an approximately linear Lineweaver-Burk plot indicated that D-leucine was transported by an active transport system for L-leucine. Vmax and ku, the first-order rate constant for the unsaturable component, for the uptake of D- and L-leucines decreased with a fall in temperature. The activation energies for Vmax and ku were in the range of 5-10 and 18-21 kcal/mol, respectively. The values for L-leucine were greater than those for D-leucine. Km for D-leucine transport increased with decreasing temperature, whereas Km for L-leucine decreased. This difference suggests that the large alkyl chains of D- and L-leucines bind to different portions of a carrier protein.     

Cell volume regulation in Ehrlich ascites tumor cells

Ehrlich cells subjected to anisoosmolar media show very rapid volume changes. In hypertonic media they shrink. In hypotonic media they swell but the rapid initial swelling is followed by a regulatory shrinkage lasting ca. 30 minutes. Cells suspended in media with identical ionic concentrations but different total osmolarity (adjusted by sucrose) were compared. These studies revealed that swollen cells adjust their volume by decreasing the amount of intracellular K⁺ and ninhydrin positive substances. Intracellular Na⁺ and ATP concentrations were unchanged. Accordingly ⁴²K⁺ flux analysis showed that the (passive) cell membrane permeability for K⁺ is increased to a minor degree and the Na⁺ permeability unaffected. The increased K⁺ permeability could not be correlated to an increase in ⁴⁵Ca²⁺ influx.     

Retention of calcium by mitochondria isolated from Ehrlich ascites tumor cells

Tightly coupled mitochondria isolated from Ehrlich ascites tumor cells accumulate and retain high concentrations of Ca²⁺ in the presence of ATP for periods up to at least 20 min at 25 °C. The presence of inorganic phosphate up to 20 mm does not prevent such Ca²⁺ retention. The tumor mitochondria accumulate Ca²⁺ in the presence of succinate as an energy source but lose the Ca²⁺ after 1–2 min. Addition of ATP (K_m approx 1 mm) to the incubation medium after Ca²⁺ release, induces reaccumulation of the ion. Thus, the ability of the tumor mitochondria to retain Ca²⁺ differs markedly from that of rat liver mitochondria and is seen as being of potential biological significance to the unique metabolic behavior of the ascites tumor cells.     

Self-inhibition of chloride transport in Ehrlich ascites tumor cells

Previous studies have shown that mediated Cl- transport which occurs by at least two processes (Cl- -dependent cation cotransport and Cl- self-exchange) becomes progressively inhibited when extracellular Cl- exceeds about 60 mM (Hoffmann et al., 1979). To account for this type of kinetic behavior, that is, self-inhibition, an anion transport system possessing two sites, a high affinity transport site and a lower affinity modifier site is suggested (Dalmark, 1976). In the present experiments we have attempted to determine which of the mediated transport pathways is susceptible to self-inhibition by studying the dependence of the steady state Cl- flux on the extracellular Cl- concentration and how DIDS, an inhibitor of Cl- self-exchange, and H + affect this relationship. Addition of DIDS to Ehrlich cells results in inhibition of Cl- transport at every Cl- concentration tested (40-150 mM). Moreover, the Cl- flux/Cl- concentration relationship no longer exhibits self-inhibition, suggesting that this phenomenon is a characteristic of the Cl- self-exchanger rather than of the Cl- -dependent cation cotransport system. Lowering the extracellular pH (pHo) from 7.35 to 5.30 stimulates Cl- transport by a process that saturates with respect to [H +]. Half-maximal stimulation occurs at pHo 6.34. A comparison of the kinetic parameters, Ks and Jmax, calculated from the ascending limb of the Cl- flux/Cl- concentration curve at pHo 7.30 to those at pHo 5.50 show that the values for Ks are almost identical (23.6 mM and 21.3 mM, respectively), while the values for Jmax [22.2 mEq/Kg dry wt) X min] differ by only 15%. This finding along with the observation that DIDS completely blocks H + stimulation of Cl- transport is compatible with the suggestion that H + interact at the modifer site of the Cl- self-exchanger and thereby prevents self-inhibition.