Fibroblasts and macrophages of mice with the Chediak-Higashi-like syndrome have microtubules and actin cables.

Cells of the beige mouse contain abnormally large lysosomes and show enhanced capping of concanavalin A. It has been suggested that these phenomena may be secondary to a defect in microtubule polymerization. We have examined the cytoskeleton of beige mouse cells by indirect immunofluorescence and find the number and distribution of microtubules and actin cables to be indistinguishable from those of normal control cells.

Interaction of BCG-activated macrophages with neoplastic and nonneoplastic cell lines in vitro : quantitation of the cytotoxic reaction by release of tritiated thymidine from prelabeled target cells.

Peritoneal cells from mice infected ip with Mycobacterium bovis, strain BCG, were cytotoxic to syngeneic tumor cells in vitro. Cytotoxicity was estimated by measurement of release of tritiated-thymidine (3-H-TDR) from prelabeled target cells. The cell responsible for tumor cytotoxicity was the macrophage. Macrophages from uninfected mice or from oil-, starch-, or thioglycollate-induced peritoneal exudates had little effect on labeled tumor monolayers. Tumoricidal macrophages were present at 3-7 days and persisted through 6 weeks after a single BCG injection. Two neoplastic/nonneoplastic cell-line pairs, all four of the cell lines derived from a cloned syngeneic embryo cell line, were used as target cells for BCG-activated macrophages. Both tumor cell lines released significantly more 3-H-TDR than did the two nonneoplastic lines. In a mixed neoplastic/nonneoplastic target cell population, BCG-activated macrophages selectively destroyed the neoplastic cells; nonneoplastic cells were not affected as "innocent bystanders".

Deceased numbers of spindle and cytoplasmic microtubules in hamster embryo cells treated with a carcinogen, diethylstilbestrol.

Diethylstilbestrol (DES) has been shown to induce neoplastic transformation in the absence of measurable mutations at specific loci in Syrian hamster embryo cells. It has been proposed that DES induces cell transformation via the production of aneuploidy. In the present study we document that concentrations of DES that cause aneuploidy also produce abnormal or arrested mitotic spindles. Thus, DES may disrupt spindle microtubules and produce aneuploidy that results in disordered gene expression and eventual neoplastic transformation.

Correlation of cytotoxicity and mitotic spindle dissolution by vinblastine in mammalian cells.

In two hamster cell lines that differed 100-fold in their vinblastine sensitivity, dissolution of the mitotic spindle by vinblastine in living cells correlated with cytotoxicity from vinblastine expressed as suppression of colony formation. The effect on the spindle apparatus occurred in 30 sec or less and thus provides a rapid assay for determining the cytotoxic effects of the Vinca alkaloids, as well as the potential for quantitative assay of solutions of Vinca derivatives.

Microtubule changes and cytotoxicity in leukemic cell lines treated with taxol.

Taxol, a diterpenoid plant product that enhances the polymerization of tubulin, is currently entering clinical trials in the treatment of human leukemia. In order to develop an in vitro assay to predict tumor sensitivity to taxol, human leukemic cell lines were exposed to clinically achievable concentrations of taxol for relevant exposure periods. Changes in microtubules visualized by indirect immunofluorescence were compared to drug sensitivity measured by a clonogenic assay. Taxol produced either multiple mitotic asters in G2/M or microtubule bundling throughout the cell cycle. In cells that were relatively resistant to taxol, microtubule bundling was reversible while microtubule bundling in relatively sensitive cells persisted in the presence or absence of taxol. In contrast, aster formation was unrelated to cytotoxicity in any cell line. In the future, these microtubule effects may be useful in predicting the chemotherapeutic efficacy of taxol.

Programmed death of nonproliferating androgen-independent prostatic cancer cells.

Androgen ablation induces an energy-dependent process of programmed death in nonproliferating androgen-dependent prostatic cancer cells which involves fragmentation of genomic DNA into nucleosomal oligomers catalyzed by nuclear Ca2+, Mg(2+)-dependent endonuclease enzymes activated following a sustained elevation in intracellular free Ca2+ (Cai). In contrast, androgen-independent prostatic cancer cells are not induced to undergo such programmed cell death by androgen ablation. One explanation for the inability of androgen ablation to induce programmed death of androgen-independent prostatic cancer cells is that such ablation does not result in a sustained elevation in Cai in these cells. This raises the issue of whether androgen-independent prostatic cancer cells can be induced to undergo programmed death if an elevation in the Cai is sufficiently sustained by nonhormonal means. To test this possibility, androgen-independent, highly metastatic Dunning R-3327 AT-3 rat prostatic cancer cells were chronically exposed in vitro to the calcium ionophore ionomycin to sustain an elevation in their Cai. These studies demonstrated that an elevation of Cai as small as only 3-6-fold above baseline can induce the death of these cells if sustained for greater than 12 h. Temporal analysis demonstrated that the death of these cells does not require cell proliferation and involves Ca(2+)-induced fragmentation of genomic DNA into nucleosome-sized pieces as the commitment step in this process. These results demonstrate that even nonproliferating androgen-independent prostatic cancer cells can be induced to undergo programmed cell death if a modest elevation in the Cai is sustained for a sufficient time. These observations identify Cai as a potential target for therapy for androgen-independent prostatic cancer cells.

Differences in the regulation of intracellular calcium in normal and neoplastic keratinocytes are not caused by ras gene mutations.

The development of resistance to terminal differentiation is an early event in epidermal neoplasia. Altered differentiation can be detected in vitro since normal epidermal cells are induced to differentiate in medium with Ca2+ greater than 0.1 mM while neoplastic epidermal cells and keratinocytes transduced with a v-rasHa gene are resistant to Ca2+. In normal epidermal cells, the elevation of extracellular Ca2+ (Cao) from 0.05 to 1.2 mM causes a biphasic intracellular Ca2+ (Cai) response in which a transient (10 min) peak of 4-5-fold over basal values is followed by a sustained (greater than 24 h) 2-fold increase in steady-state Cai. The transient peak in Cai is dependent on a serum component and independent of Cao, while the sustained plateau is directly dependent on Cao. The transient peak responding to a serum factor is lost in normal cells after 24 h in 1.2 mM Ca2+, a time when these cells are differentiating. Two neoplastic keratinocyte cell lines, SP-1 and 308, which produce benign tumors in vivo, also have a biphasic Cai response to an increase in Cao. In these cells, the transient peak is also serum dependent and amplified to 10-fold over basal values. However, the plateau value is not sustained and returns to basal values by 8 h, independent of Cao. Furthermore, 308 cells remain sensitive to the serum-induced Cai transient after 24 h in 1.2 mM Ca2+. To determine whether the activating c-rasHa mutation in 308 and SP-1 cells was responsible for the altered Cai regulation, a v-rasHa gene was introduced into normal keratinocytes by a defective retrovirus. This also produces the papilloma phenotype in vivo. Recipient cells were resistant to Ca(2+)-induced terminal differentiation although they did not proliferate in 1.2 mM Ca2+. The Cai profile in response to 1.2 mM Ca2+ was identical in normal and v-rasHa keratinocytes, and these cells lost the serum-induced transient Cai peak after 24 h. Thus, the activation of the c-rasHa gene in 308 or SP-1 cells is probably not solely responsible for the altered Cai response in neoplastic cell lines. Sustained physiological elevation of Cai may be relevant to the loss of proliferative potential in both normal and v-rasHa keratinocytes in 1.2 mM Ca2+. In addition, v-rasHa-mediated or activated c-rasHa-mediated changes in a complementary pathway may contribute to the block in terminal differentiation in neoplastic cells.

Colony morphology and growth in agarose as tests for spontaneous neoplastic transformation in vitro.

Adherent fibroblast-like cells from paired lines, one non-neoplastic and the other "spontaneously" transformed neoplastic, were compared in simultaneous in vivo and in vitro assays. The in vivo assay was the i.m. implantation of 10(6) or 10(7) cells in irradiated syngeneic animals, and the two in vitro assays were the evaluation of colony morphology on plastic and the enumeration of colony growth in semisolid agarose. The percentage of colonies diagnosed from their morphology as neoplastic correlated with tumorigenicity as follows: 100% always indicated a tumorigenic cell population with tumor latent periods from 6 to 230 days and tumor incidence from 40 to 100%; 0% always indicated a nontumorigenic cell population; 1 to 32% indicated either a tumorigenic cell line with long tumor latent period (218 days) with 70% tumor incidence or a nontumorigenic cell line. Growth in agarose, as measured by colony number and size, correlated with tumorigenicity as follows: nontumorigenic cell lines produced no colonies; tumorigenic cell lines produced colonies, but not always larger than 0.1 mm in diameter. The number of size or colonies did not correlate with the tumor latent period or tumor incidence. Therefore, both in vitro tests were reliable qualitative assays of spontaneous neoplastic transformation, but they did not correlate directly with the tumor incidence or mean tumor latent period. The relative success of the agarose assay emphasizes the importance of decreased anchorage dependence for progressive growth of injected cells as a malignant neoplasm in vivo.

Population density as a factor in the evolution of neoplastic cell lines.

The influence of population density in the progression from the nonneoplastic to the neoplastic state has been reassessed. Two twice-cloned, nonneoplastic mouse lines, NCTC 7914 and 7915, were transferred each 3 to 4 days at inoculum sizes selected to minimize or maximize cell-cell contact, 1 X 10(5) or 4 X 10(5) cells/T-15, respectively. As tested by in vivo assay, the regime designed to minimize cell-cell contact did not reproducibly delay transformation, and tumor production was observed in all lines, irrespective of inoculum size. Also, results of tumorigenesis assays correlated with blind evaluation of morphological and cytological alterations, growth in agarose, and susceptibility to killing by activated macrophages. Generally higher saturation densities were seen as a function of period in culture, and no significant differences in glucose utilization or lactic acid production were observed between nonneoplastic and neoplastic cell populations.

Phase I and pharmacodynamic study of taxol in refractory acute leukemias.

Taxol, a novel antimicrotubule agent that enhances tubulin polymerization and microtubule stability, was administered to adults with refractory leukemias as a 24-h i.v. infusion in a Phase I study. The primary objectives were to determine the maximum tolerated dose of taxol administered on this schedule to patients with acute leukemias and describe the nonhematological toxicities which became dose limiting. The starting dose, 200 mg/m2, was based on the maximum tolerated dose in solid tumor trials, in which myelosuppression precluded dose escalation. Seventeen patients received 28 evaluable courses at 200, 250, 315, and 390 mg/m2. Severe mucositis limited further dose escalation. Other nonhematological effects included peripheral neuropathy, alopecia, myalgias, arthralgias, nausea, vomiting, diarrhea, and an acute pulmonary reaction that was presumptively due to taxol's Cremophor vehicle. Mean peak taxol plasma concentrations at all dose levels were in the range of concentrations that were previously demonstrated to induce microtubule bundles, a morphological effect associated with cytotoxicity, in leukemia cells in vitro. Pretreatment blasts from 12 patients were incubated with taxol ex vivo. Taxol-induced microtubule bundles were apparent in the blasts of eight patients who also had cytoreduction of tumor, and sensitivity to bundle formation was related to the magnitude of antitumor activity. In contrast, taxol did not induce microtubule bundles ex vivo in the blasts of the other four total nonresponders. Based on this study, the maximum tolerated doses and recommended Phase II doses for taxol, limited by nonhematological toxicity and administered as a 24-h i.v. infusion to patients with refractory leukemias, are 390 and 315 mg/m2. Phase II trials at these myelosuppressive doses are required to determine taxol's activity in the treatment of leukemias. In addition, further evaluation of microtubule bundle formation ex vivo in Phase II studies is necessary to determine the ultimate utility of this assay in assessing tumor sensitivity to taxol.

Cell shape and increased free cytosolic calcium [Ca2+]i induced by growth factors.

Growth factors stimulate DNA synthesis of neoplastic cells but not of non-neoplastic cells in suspension cultures. Similarly, growth ceases in dense monolayers of non-neoplastic cells, while crowded neoplastic cells continue to grow. The mechanism of these important phenotypic changes is unknown; the block in growth stimulation could occur in early events of signal transduction at the plasma membrane or in a late step in the final steps of gene activation and induction of DNA synthesis. One particular early intracellular event, [Ca2+]i increases, is in fact necessary for the induction of DNA synthesis in attached non-neoplastic Balb/c 3T3 cells stimulated by platelet-derived growth factor (PDGF). We therefore used digital image analysis of intracellular Fura-2 fluorescence to determine whether PDGF can stimulate [Ca2+]i transients in suspension or in dense monolayer cultures of Balb/c 3T3 cells. In dense cells (greater than 8 x 10(4) cells/cm2) the basal [Ca2+]i and [Ca2+]i response to PDGF stimulation were both lower than those in sparser, more spread cells. PDGF also did not release internal stores of Ca2+ or produce Ca2+ influx in completely suspended cells. Remarkably, attachment alone, with minimal cell spreading, was enough to reinitiate the entire early signalling mechanism stimulated by PDGF. Thus, a block in PDGF-induced [Ca2+]i increases may contribute to the inability of PDGF to stimulate DNA synthesis in suspended non-neoplastic cells. This early block in signal transduction must be abrogated in neoplastic cells growing in suspension and dense monolayer cultures.

Distribution of intracellular free calcium in quiescent BALB/c 3T3 cells stimulated by platelet-derived growth factor.

A digital imaging microscope and fluorescent Ca(2+)-sensitive probe (Fura 2) were used to study the spatial location and time course of increases in free intracellular calcium (Cai) induced by platelet-derived growth factor (PDGF). Microinjection of Fura 2 acid avoided problems of incomplete deesterification of Fura 2-acetoxymethyl ester (Fura 2/AM) and dye localization in cellular organelles. PDGF stimulated a rapid increase in Cai (up to 8-fold increase) in both the nucleus and the cytoplasm in approximately half of the quiescent BALB/c 3T3 cells. Cai changes were both spatially and temporally heterogeneous, the latter including both transient (1-2 min) and prolonged increases (greater than 5 min) in the same cell. PDGF stimulated mitogenesis and Cai increases in approximately the same percentage of cells. Moreover, large intracellular concentrations of a Ca2+ buffer (Quin 2) inhibited both Cai increases and mitogenesis stimulated by PDGF. Thus, Ca2+ increases in the nuclear and/or cytosolic compartments appear to be required for the stimulation of mitogenesis by polypeptide growth factors such as PDGF.

Cytosolic free calcium and DNA synthesis in BALB/c 3T3 cells: aequorin luminescence studies.

In order to investigate how growth factors stimulate DNA synthesis, we studied the relationship between early increases in the intracellular concentration of free calcium (Cai) and early mitogenic events (competence) in quiescent BALB/c 3T3 cells. Cai was measured by aequorin luminescence, and DNA synthesis was quantitated by autoradiography. While both serum (2%) and platelet-derived growth factor (2 U/ml) stimulated large increases in Cai without inducing competence, fibroblast growth factor (100-400 ng/ml) produced a large number of competent cells without causing a detectable increase in Cai. Thus, there was no correlation between the magnitude of Cai increase and subsequent competence for DNA synthesis.

Phase I study of twice-weekly gemcitabine and concurrent thoracic radiation for patients with locally advanced non-small-cell lung cancer.

PURPOSE: To determine the maximum tolerated dose (MTD) and dose-limiting toxicity of twice-weekly gemcitabine and concurrent thoracic radiation in patients with Stage IIIa/IIIb non-small-cell lung cancer (NSCLC). METHODS AND MATERIALS: Seventeen patients with histologically confirmed Stage IIIa and IIIb NSCLC were studied. Gemcitabine was administered via a 30-min i.v. infusion twice weekly for 6 weeks concurrent with 60 Gy of thoracic radiation. Gemcitabine, starting at a twice-weekly dose of 10 mg/m2 (20 mg/m2/week), was escalated in 10-15 mg/m2 increments in successive cohorts of 3 to 6 patients until dose-limiting toxicity was observed. RESULTS: Of the 17 patients entered, 16 were evaluable for toxicity. The dose-limiting toxicity at 50 mg/m2 given twice weekly (100 mg/m2/week) was Grade 3 pneumonitis observed in 1 patient, Grade 3 pulmonary fibrosis in a second patient, and Grade 4 esophagitis observed in two additional patients. Twice-weekly gemcitabine at a dose of 35 mg/m2 was determined to be the MTD. The overall response rate for the 16 evaluable patients was 88%. The median survival for the entire group is 16.0 months. CONCLUSIONS: The MTD of twice-weekly gemcitabine is 35 mg/m2 (70 mg/m2/week) given with thoracic radiation. A Phase II study within the Cancer and Leukemia Group B to ascertain the potential efficacy of this treatment regimen is in development.

Signal transduction for proliferation and differentiation in keratinocytes.

In mouse and human epidermis, the Ca2+ environment of the basal cell layer is substantially below serum Ca2+, while that of the granular cell layer is unusually high. Reduction of extracellular Ca2+ concentration (Cao) in the medium of keratinocyte cultures maintains a basal cell phenotype while serum Ca2+ concentrations induce terminal differentiation. Measurements of intracellular Ca2+ (Cai) by the use of Fura 2 and digital imaging technology reveal that Cai increases 10-20-fold in response to an increase in Cao and remains elevated. Concomitant with the rise in Cai is an increase in the metabolism of phosphatidylinositol (PI) to yield inositol phosphates and diacylglycerol. PI metabolism is also stimulated by calcium ionophores suggesting that a rise in Cai is directly responsible. The consequent increase in diacylglycerol and Cai would activate protein kinase C, an event known to trigger epidermal differentiation. Specific Cao and Cai determine the expression of individual markers of keratinocyte differentiation in vitro. These findings may account for the importance of the Ca2+ gradient for maintaining regulated growth and differentiation of the epidermis in vivo.

Centriole ciliation and cell cycle variability during G1 phase of BALB/c 3T3 cells.

Although variability in the duration of the cell cycle is thought to reflect growth-regulatory processes that control cell cycle progression, the precise timing of the variable period within the G1 phase of the cell cycle has not been defined. In particular, the timing of cell cycle variability in relation to the cell's commitment (R point) to the initiation of DNA synthesis remains controversial. In order to investigate cell cycle variability, indirect immunofluorescence was used to measure the formation of the primary cilium as a possible marker of G1 events in both stimulated quiescent and exponentially growing cells. The primary cilium, an internal "9 + 0" nonmotile structure formed by one of the interphase centrioles, was first detected in postmitotic BALB/c 3T3 cells 5 hr before the initiation of DNA synthesis, an interval similar to that for the reassembly of the primary cilium in serum-stimulated quiescent fibroblasts. This similarity in the timing of ciliation suggests that serum-stimulated quiescent cells reenter the cell cycle in early G1 and recapitulate much of G1. Moreover, the rate of cilia formation in both postmitotic and serum-stimulated quiescent cells was identical to the rate of DNA synthesis initiation. Thus, cell cycle variability occurs before ciliation in both stimulated quiescent and exponentially growing cells. Furthermore, since ciliation also precedes the R point, variability in the centriole cycle occurs before the R point and thus may reflect processes controlling the cell's commitment to the initiation of DNA synthesis.

Tubulin and actin in paired nonneoplastic and spontaneously transformed neoplastic cell lines in vitro: fluorescent antibody studies.

Pairs of nonneoplastic and spontaneously transformed neoplastic cells were derived from rat, mouse and hamster embryos. The neoplastic cells of each pair had poorly spread cellular morphology, grew in agarose in vitro and produced invasive sarcomas in vivo; the nonneoplastic cells exhibited none of these properties. The distribution of microtubules and microfilament bundles (stress fibers or actin cables) was examined in five such paired lines and in 3T3 and SV40-transformed 3T3 cells by indirect immunofluorescent microscopy of fixed cells treated with rabbit antibody prepared against bovine brain tubulin or guinea pig smooth muscle actin, respectively. Actin cables in all the neoplastic cells appeared thinner and more sparse than in the paired nonneoplastic cells. These differences were also observed in living cells with polarization microscopy. In contrast, microtubules appeared similar in neoplastic and nonneoplastic cells, both in areas of thin peripheral lamellar cytoplasm which allowed a clear visualization of fine, curving microtubules and in regions of thick, central endoplasm which obsecured individual microtubules. In fact, the main morphological difference between neoplastic and nonneoplastic cells was the relative amount of lamellar cytoplasm or endoplasm, rather than the appearance of microtubles in either region. Thus the distinctive growth properties and retracted cellular morphology of neoplastic cells in this study did not correlate with decreased or disorganized microtubules, but with thin and sparse actin cables.