Cell kill kinetics and cell cycle effects of taxol on human and hamster ovarian cell lines.

Taxol is a clinically active anticancer drug, which exerts its cytotoxicity by the unique mechanism of polymerizing tubulin monomers into microtubules and stabilizing microtubules. Our studies with ovarian (hamster CHO and human A2780) cells showed that taxol is a phase-specific agent that is much more cytotoxic to mitotic cells than interphase cells. First, the dose-survival pattern of taxol resembled that of other phase-specific agents, in which cell-kill reached a plateau at a certain concentration. This suggests that the asynchronous cell population consists of a taxol-sensitive (presumably mitotic) fraction and a taxol-resistant fraction. Second, the cells were more responsive to increased exposure time than to increased dose above the plateau concentration. Third, in both asynchronous and synchronous cultures taxol was much more cytotoxic to mitotic than interphase (G1, S and G2) cells. Fourth, the taxol concentration needed to kill cells corresponded to the dose needed to block cells in mitosis. Although taxol blocked cells in mitosis, the mitotic block was of short duration. Cells escaped the mitotic block, without cytokinesis, and entered the next round of DNA synthesis to form multinucleated polyploid cells. Taxol was 15- to 25-fold more toxic to A2780 (human ovarian carcinoma) cells compared to CHO cells. This difference in sensitivity correlated with a higher intracellular taxol concentration in A2780 as compared to CHO as determined by either an ELISA assay or by [H3]-taxol uptake.

Prevention of tilorone developmental toxicity with progesterone.

The immunomodulator tilorone hydrochloride was administered (gastric intubation) once to time-pregnant Upj:TUC(SD)spf (Sprague-Dawley) rats in four experiments. In experiment 1, tilorone (250 or 500 mg/kg) was administered on day 10 of gestation. The dams were killed 4 or 72 hr after dosing. Interferon-like activity and drug levels were determined in maternal blood, spleen, and thymus, as well as in the embryos. In experiment 2, the test groups received progesterone (2 mg/kg), or tilorone (200 or 400 mg/kg), or progesterone and tilorone. The dams from each group were killed 24 or 48 hr after receiving tilorone. Experiment 3 was similar to experiment 2, except that the dams were killed on gestation day 20. In experiment 4, tilorone (400 mg/kg) was administered on gestation day 17, 18, or 19, and the dams were killed 24 hr after dosing or on gestation day 20. In all four experiments, tilorone-related maternal toxicity (regardless of whether progesterone also was administered) was observed, as characterized by marked decreases in weight gain, the occurrence of clinical signs, and in experiment 1 by decreased thymus weights, 72 hr post-dosing. Dose-related increases in the mean number of dead embryos and in serum interferon titers occurred 72 hr postdosing. In experiment 2, there was an increase in the number of dams in the 400-mg/kg (tilorone only) group with dead embryos only, 24 hr postdosing; similar results occurred in both the 200- and 400-mg/kg groups, 48 hr postdosing. However, in the groups that also received progesterone, a partial prevention of such embryolethality was evident. In experiment 3, embryotoxicity again was observed in both tilorone-treated groups, whereas several of the dams that were also given progesterone through day 19 of gestation experienced at least a partial prevention of the embryolethal effects of tilorone. In experiment 4, no fetotoxicity was observed despite the severe maternal toxicity evident.

Soluble CD4-PE40 is cytotoxic for a transfected mammalian cell line stably expressing the envelope protein of human immunodeficiency virus (HIV-1), and cytotoxicity is variably inhibited by the sera of HIV-1-infected patients.

Sera were obtained from 50 individuals infected with human immunodeficiency virus type 1 or from HIV-1-uninfected individuals before or after vaccination with recombinant gp160. These sera were evaluated for activity antagonistic to the cell-killing activity of the chimeric Pseudomonas exotoxin hybrid protein, sCD4-PE40. For these studies, Chinese hamster ovary (CHO) cells were transfected with a chimeric plasmid encoding the tat, rev, and envelope genes of HIV-1 and a cell line was selected for stable expression of the envelope glycoproteins at the cell surface (CHO-env). Cytotoxicity of sCD4-PE40 for CHO-env in the presence or absence of added human serum was quantitated spectrophometrically following enzymatic reduction of a tetrazolium bromide within the mitochondria of viable cells (MTT assay). Several HIV+ sera inhibited the cytotoxic activity of sCD4-PE40; the antagonist had properties consistent with those of immunoglobulins in that it was heat stable, absorbed by protein A, and reversible by increasing the concentration of sCD4-PE40. Of 15 HIV+ sera which strongly reacted with gp120, 11 (73%) also potently inhibited sCD4-PE40 cytotoxicity, and cytotoxicity was inhibited by sera from some HIV- individuals after, but not before, immunization with gp160. These data suggested a role for antibody to gp120 in the antagonistic activity. However, not all sera with antibody to gp120 antagonized sCD4-PE40 cytotoxicity and high levels of antagonist activity were frequently (40%) found in HIV+ sera lacking immunoblot-detectable antibody to gp120, or antibody to either CD4 or PE40. Grouping of the HIV+ sera according to the patients' absolute number of CD4+ cells revealed that the degree of inhibition of sCD4-PE40 cytotoxicity approached a Gaussian distribution, suggesting that persons with CD4+ cell counts between 200 and 700/mm3 may be more likely to possess significant levels of serum antagonist. This data have implications for the clinical development of sCD4-PE40 or other sCD4-based therapeutics in the management of HIV-1 infection.

Secretion of active truncated CD4 into Escherichia coli periplasm.

A truncated molecule containing the first 183 amino acid residues of the HIV-1 receptor, CD4, was made by periplasmic secretion in Escherichia coli. The signal sequence from the E. coli proteins OmpA, PhoA, or OmpF was fused to the truncated CD4, under the control of either the trp or the lac promoter. The processed material secreted into the periplasm reacted with monoclonal antibodies and exhibited binding activity to the HIV-1 envelope protein gp120. Not all of the processed product was recovered in the periplasm by osmotic shock, suggesting that either the material aggregated in the periplasm or, during secretion, the molecule assumed some transient conformation that interfered with its translocation across the inner membrane. A mutation in prlA (a gene involved in secretion) increased the level of processing, suggesting that secretion of a heterologous protein in E. coli can be optimized by manipulating the host secretion apparatus.

A rapid, quantitative bioassay based on the human immunodeficiency virus trans-activator.

We constructed a human immunodeficiency virus (HIV) trans-activator cDNA (tat) encoding the N-terminal 76 amino acids of the viral trans-activator followed by two additional amino acids (val and pro). This cDNA encoded a functional trans-activator (TAT) as shown by cotransfection into murine cells with a HIV promoter-chloramphenicol acetyltransferase DNA construct. The tat cDNA was cloned into an avian retroviral expression vector, a modified spleen necrosis virus (SNV), and high-titer infectious stocks of recombinant virus (SNV-tat) were recovered from dog cells. Hybridization analyses indicated that SNV-tat was stably propagated in these cells for months. We also prepared recombinant cells that stably carry reporter genes, either a human gene encoding a soluble CD4 receptor (sCD4) or the human preprorenin gene, under the transcriptional control of the HIV promoter. Medium obtained from these cell cultures after infection with control viruses or an SNV carrying an antisense tat contained only low background levels of sCD4 or prorenin (HRN) as determined by specific immunoassays (1-10 ng protein per 10(6) cells per ml medium). In contrast, cells infected with SNV carrying tat in the transcriptional sense orientation secreted 75 +/- 7 ng sCD4 and 73 +/- 4 ng HRN per 10(6) cells per ml medium. Moreover, these proteins were constitutively secreted at these levels during months of subculturing. The data indicate that sCD4 and HRN are secreted from these cells because of a TAT-mediated trans-activation of the HIV reporter gene DNA and/or RNA. This combination of recombinant cells, SNV-tat, and specific immunoassays provide a rapid, quantitative, and safe bioassay to seek inhibitors of TAT.

A rapid solution immunoassay to quantify binding of the human immunodeficiency virus envelope glycoprotein to soluble CD4.

We developed a particle concentration fluorescent immunoassay to quantify the binding in solution of the human immunodeficiency virus (HIV) external glycoprotein (gp120) to soluble CD4 (sCD4). The assay is rapid (1 hr), quantitative, and requires as little as 0.1 pmole of gp120 per evaluation. We find that gp120, purified from recombinant baculovirus infected insect cells, is suitable for the assay. Moreover, sCD4s obtained either from recombinant E. coli or mammalian cells, consisting of the N-terminal two domains (about 180 amino acids) as well as linked to the active regions of Pseudomonas exotoxin A, bind gp120 similarly.

Granulocyte transfusion therapy of experimental Pseudomonas pneumonia.

Pseudomonas pneumonia was produced in dogs with radiation-induced leukopenia. Treatment of this infection with either gentamicin alone or gentamicin plus daily granulocyte transfusion was compared in a randomized controlled trail. The dogs receiving granulocytes plus gentamicin survived significantly longer than those treated with gentamicin alone (P < 0.05). The Pseudomonas immunotype which was inoculated into the dogs were recovered at autopsy from none of the granulocyte-transfused dogs, whereas seven or eight of the dogs treated with gentamicin alone had the inoculated Pseudomonas immunotype in the area of induced pneumonia at autopsy. As measured by the limulus test, the granulocyte-transfused dogs also did not have endotoxemia as frequently as the dogs given only gentamicin (P < 0.05). This controlled study establishes that transfused granulocytes can favorably alter the course of experimental Pseudomonas pneumonia and suggests that granulocyte transfusion may be a useful therapy in serious bacterial infections of leukopenic subjects.