Clonal Growth Assays Research Articles

Leptomycin B stabilizes and activates p53 in primary prostatic epithelial cells and induces apoptosis in the LNCaP cell line.

Previous studies showed that primary cultures of normal and malignant human prostatic epithelial cells are defective in their ability to upregulate the tumor suppressor protein p53 in response to DNA damage. This dysfunctional regulation of p53 may be relevant to both the high incidence of prostate cancer and its resistance to chemotherapy. Leptomycin B (LMB) has recently been found to increase the protein level and transcriptional activity of p53 by interfering with nucleocytoplasmic export and subsequent degradation by the proteasome. We investigated the ability of LMB to activate p53 in prostatic epithelial cells. Primary cultures and the cell lines LNCaP and DU 145 were treated with LMB. p53 protein was evaluated in Western blots and by immunocytochemistry. Induction of downstream targets of p53 was evaluated in Western and Northern blots. Growth inhibition, cell cycle arrest, and apoptosis in response to LMB were measured in clonal growth assays, by flow cytometry, and by Hoescht/propidium iodide staining, respectively. Treatment of prostatic epithelial cells with LMB led to post-translational stabilization of p53, activation of downstream target genes, and induction of cell cycle arrest in primary cultures and apoptosis in LNCaP (with wild-type p53) but not DU 145 (with mutant p53) cells. p53 in primary cultures of normal and malignant prostate cells, although dysfunctional in that it is not responsive to DNA damage, is activated by LMB. The ability of LMB to stabilize p53 and induce expression of p53-responsive growth inhibitory genes may be a useful lead in the development of chemopreventive or therapeutic small molecules that can modulate p53 function in prostatic epithelial cells.

CURCUMIN PREVENTS INTRAVESICAL TUMOR IMPLANTATION OF THE MBT-2 TUMOR CELL LINE IN C3H MICE

The development of an effective nontoxic intravesical agent that may be used immediately after bladder tumor resection to prevent the implantation of tumor cells would be a significant clinical advancement. We report the cytotoxic effects of curcumin on bladder tumor cell lines as well as its effects on the intravesical implantation of tumor cells in C3H mice. UMUC human and MBT-2 mouse bladder cancer lines were incubated with 0 to 100 microM. curcumin in dimethyl sulfoxide for 30 minutes and cell viability was determined by clonal assay. Additional culture dishes were incubated with curcumin and processed for electron microscopy. Using the C3H mice and the MBT2 tumor lines the effects of intravesical curcumin on tumor implantation after bladder injury was studied. The 10 group 1 mice served as nontreatment controls. In the 18 group 2 mice 30 minutes after tumor cell implantation 100 microM. curcumin in 0.1% dimethyl sulfoxide were instilled intravesically for 30 minutes. The 15 group 3 mice served as treatment controls with 0.1% dimethyl sulfoxide or culture medium instilled intravesically for 30 minutes. Animals were sacrificed 7 to 10 days after treatment and the bladder was subjected to histological analysis for tumor. At the 100 microM. dose curcumin was completely lethal to the 2 cell lines on clonal growth assay. Electron microscopy revealed apoptotic bodies after curcumin administration. The tumor implantation rate was 16.7% (3 of 18 mice) in curcumin treated bladders and 73% (11 of 15) in the vehicle control group. At the 100 microm. concentration curcumin is a potent cytotoxic agent against the MBT and UMUC bladder tumor cell lines. In addition, curcumin effectively inhibits tumor implantation and growth in this murine bladder tumor model.

Autocrine Transforming Growth Factor α Provides a Growth Advantage to Malignant Cells by Facilitating Re-entry into the Cell Cycle from Suboptimal Growth States

CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor alpha (TGF-alpha) was ectopically expressed by stable transfection of a TGF-alpha cDNA under repressible tetracycline control. The TGF-alpha-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-alpha transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-alpha-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to re-enter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-alpha-transfected cells indicate that autocrine TGF-alpha functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-alpha was associated with increased malignant properties as TGF-alpha transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-alpha expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-alpha. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-alpha.

Basic FGF, EGF, and PDGF modify TGFβ-induction of smooth muscle cell phenotype in human prostatic stromal cells

We investigated the ability of a variety of growth factors to regulate the differentiation of prostatic fibroblasts into smooth muscle cells. Smooth muscle actin levels were monitored by immunoblot analysis and immunocytochemistry. Proliferation was measured in clonal growth assays and by cell counts. We determined that TGFbeta inhibited proliferation and induced smooth muscle differentiation of stromal cells derived from prostatic adenocarcinomas, as we previously reported for cells derived from the normal peripheral zone. Basic FGF, EGF, TGFalpha, and PDGF, but not IGF, retinoic acid, 1,25-dihydroxyvitamin D3, or androgen, attenuated induction of differentiation by TGFbeta, by a mechanism apparently unrelated to proliferation. Regulation of growth and differentiation occurs equivalently in prostatic stromal cells derived from adenocarcinomas and normal peripheral zone. TGFbeta is a potent inducer of the smooth muscle phenotype. Basic FGF, EGF and/or TGFalpha, and PDGF attenuate TGFbeta's activity, and promote a fibroblastic phenotype. Our studies provide an in vitro model system in which fibroblastic or smooth muscle cells can be promoted, maintained, and investigated in a defined manner. The results suggest that the ratio of fibroblasts to smooth muscle cells in the stroma reflects the relative levels of growth factors, which may be altered in diseased states.

A protein-free solution as replacement for serum in trypsinizationprotocols for anchorage-dependent cells

Many animal cell culture media require serum as a component necessary for supporting the growth and survival of the culture. However, serum is of unknown composition, which may vary from lot to lot, and is very protein-rich. Therefore, serum-free media have been developed for the investigation of nutritional demands of animal cells in vitro and the production of therapeutic proteins with animal cells. Despite the fact that there are many published serum-free media for anchorage-dependent animal cells, only little work has been done on the development of serum- and protein-free subcultivation techniques. However, the serum-free subcultivation of animal cells is often a cumbersome attempt resulting in clumping and loss of the major part of the culture. Here we describe a new subcultivation technique for anchorage-dependent cells. Anchorage-dependent cells were first detached from the culture surface by trypsinization, suspended in phosphate buffered saline, 0.02% (w/v) EDTA, 75 mg/l phenol red, and 1% (w/v) pluronic F-68, and centrifuged to separate the cells from trypsin. The use of a trypsin inhibitor was not necessary. Cells were then resuspended in medium and seeded into new tissue culture vessels. No interference of the subcultivation process with the viability of the cells was observed as judged from clonal growth assays.

Transgenic expression of PML/RARalpha impairs myelopoiesis.

The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene (PML) on chromosome 15 with the retinoic acid receptor alpha (RARalpha) on chromosome 17. This yields a fusion transcript, PML/RARalpha, a transcription factor with reported dominant negative functions in the absence of hormone. Clinical remissions induced with all-trans retinoic acid (RA) treatment in acute promyelocytic leukemia are linked to PML/RARalpha expression in leukemic cells. To evaluate the PML/RARalpha role in myelopoiesis, transgenic mice expressing PML/RARalpha were engineered. A full-length PML/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice. Expression was confirmed in the bone marrow with a reverse transcription PCR assay. Basal total white blood cell and granulocyte counts did not appreciably differ between PML/RARalpha transgenic and control mice. Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice. However, in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors, especially in those responding to granulocyte/ macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition. Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation. Following irradiation, PML/RARalpha transgenic mice, as compared with controls, more rapidly depressed peripheral white blood cell and granulocyte counts. As expected, nearly all control mice (94.4%) survived irradiation, yet this irradiation was lethal to 45.8% of PML/RARalpha transgenic mice. Lethality was associated with more severe leukopenia in transgenic versus control mice. Retinoic acid treatment of irradiated PML/RARalpha mice enhanced granulocyte recovery. These data suggest that abnormal myelopoiesis due to PML/RARalpha expression is an early event in oncogenic transformation.

Limbal Epithelium Is More Resistant to 5-Fluorouracil Toxicity Than Corneal Epithelium

There is increasing evidence indicating that the limbal epithelium contains slow-cycling stem cells, whereas both limbal and corneal epithelia contain rapid-cycling transit amplifying cells. 5-Fluorouracil (5-FU), an antimetabolite used in glaucoma filtering procedures, is reported to inhibit rapid-cycling cells. After a 1-day subconjunctival (20 mg/eye) 5-FU injection followed by a 2-day rest, continuous BrdU labeling for 5 additional days (to also label slow-cycling stem cells) revealed that the limbal labeling index was not changed, whereas the corneal labeling index was markedly suppressed (p < 0.001). Following subconjunctival 5-FU treatments, peripheral and central corneal explant cultures showed a significantly higher magnitude of reduction of epithelial outgrowth size than limbal cultures (p < 0.05). Such a resistant nature of limbal epithelium to 5-FU toxicity was further confirmed by two different single-cell clonal growth assays. Taken together, these results indicate that the limbal epithelium has more slow-cycling cells than the corneal epithelium, and these cells selectively harvested by the subconjunctival 5-FU treatment can be used for future studies of their regulatory mechanism.

Qualitätskontrolle in der Hornhautbank - eine notwendige Maßnahme?

During a period of 3 months 20% of patients who received a corneal transplant showed a prolonged postoperative swelling, of the stroma and an increased number of folds in the epithelium was prolonged. These findings were atypical because there were no complications during the operation and the preoperative examination of the donor cornea showed a normal corneal endothelium. All donor corneas were stored in organ culture before transplantation using minimal essential medium supplemented with 2% fetal calf serum (FCS). This method has been used in the university eye clinic of Hamburg since 1981. In order to establish any correlation between the used organ culture conditions and the clinical findings the different components of the culture medium were checked. We noticed that the serum batch used for organ preservation had been changed at the beginning of the above mentioned period. From April 20th to June 23rd, 28 penetrating keratoplasties were performed at the department of ophthalmology of the University Clinic of Hamburg. No complications during the surgical procedure or postoperative infection were detectable. Four weeks after transplantation all transplants showed a normal swelling. The quality of media or sera is not guaranteed by the company where the substances were purchased. We performed clonal growth assays using human or porcine corneal endothelial cells to evaluate the quality of different charges of fetal calf serum. It could be demonstrated that the acticity of the serum charge used for organ preservation during the described period had a significantly reduced activity in a clonal growth assay compared with various FCS-charges from different companies. After changing the serum batch the problems described above disappeared. We conclude that quality testing of all supplements of the organ culture medium is necessary in a cornea bank to guarantee a reliable standard of corneal preservation. A clonal growth assay is a sensitive test which is suggested to be useful for quality testing.

Effects of trace metals on mouse B16 melanoma cells in culture.

The effects of fourteen metal ions (As3+, As5+, Cd2+, Co2+, Cr3+, Cr6+, Hg2+, Li+, Mg2+, Mn2+, Ni2+, Se4+, V5+, VO2+) on the proliferation and differentiation in mouse B16 melanoma cells cultivated in vitro were analyzed. Cell number assays, melanin, and protein measurements, a 3(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide reduction test (MTT survival test), and a clonal growth assay were performed. At 10(-4)M, metal ions such as As3+, As5+, Cd2+, Cr6+, Se4+, V5+, VO2+, and, to a minor extent, Li+, Hg2+, and Co2+ significantly reduced the number of the B16 melanoma cells. For the same molar concentration, the order of the levels of cell toxicity of the metal compounds to B16 cells as measured by the MTT test was as follows: Hg2+ > Cr6+ = Cd2+ > As3+, As5+, > V5+, VO2+ > Se4+ = Ni2+ = Co2+ = Li+. An increased synthesis of melanin in B16 cells was noted after incubation with Co2+, Ni2+, Cd2+, and Li+, whereas Se4+ had, on the contrary, an inhibiting effect on melanogenesis.

Vitamin A regulates proliferation and differentiation of human prostatic epithelial cells

The response of cultured human prostatic epithelial cells to vitamin A was measured by clonal growth assay in serum-free medium. Retinoic acid at 3 nM or higher inhibited the proliferation of cell strains derived from normal, benign hyperplastic and malignant tissues, while lower levels (0.03 nM) were stimulatory. Reduced proliferation induced by retinoic acid was accompanied by a marked change in morphology, as intercellular adhesion decreased. In conjunction, the expression of keratins 8 and 18, associated with the differentiated luminal phenotype of prostatic epithelia, was increased. In post-confluent cultures, retinoic acid prevented the appearance of keratin 1, which accompanied the development of a squamous phenotype by cells maintained under these conditions. The findings of this study indicate a role for vitamin A as a modulator of the growth and differentiation of prostatic epithelial cells.

Serum-free culture of fractionated bovine bronchial epithelial cells

Procedures for the serum-free culture of a density fractionated population of bovine bronchial epithelial cells have been established. Epithelial cells dispersed by protease digestion were fractionated by density equilibrium centrifugation, followed by plating of the small basal-like population on type I collagen-coated culture dishes. Two or three passages of 1:4 split enriched for a population of actively dividing cells, which could be stored in liquid nitrogen for subsequent use. Clonal growth assays revealed optimum proliferation using a 1:1 mixture of medium RPMI 1640 and LHC-9, a medium employed for human bronchial epithelial cells. Cellular growth rate, which was 0.6 to 1.3 doublings per day depending on the cell preparation, was conveniently decreased by supplementing LHC-9 medium with less than 50% RPMI. In contrast to airway epithelial cell cultures from other species, serum stimulated the growth of bovine bronchial epithelial cells in this system. Transforming growth factor beta 1, however, inhibited growth and induced differentiation into a squamous phenotype. Also in contrast with other systems, the bovine cells were resistant to growth inhibition by 100 nM tetradecanoyl phorbol acetate or 1 microM calcium ionophore A23187. Combination of phorbol ester with ionophore decreased mitotic activity, although induction of squamous morphology was not observed. Therefore, growth inhibition and squamous differentiation were not tightly coupled in this system. Finally, biologically synthesized matrix deposited by these cells stimulated growth rate. This culture system will therefore be useful in assessing the activities of both soluble and matrix-associated factors in the absence of serum.

Taurine behaves as an osmolyte in Madin-Darby canine kidney cells. Protection by polarized, regulated transport of taurine.

Using a clonal growth assay, we demonstrated that taurine, a nonperturbing osmolyte accumulated in kidney medulla, brain, and some other tissues of hypertonic experimental animals can function as a nonperturbing osmolyte in Madin-Darby canine kidney (MDCK) cells. The taurine content of hypertonic MDCK cells is twice that of isotonic MDCK cells (isotonic 160 nmol/mg protein; hypertonic 320 nmol/mg protein). Therefore we studied taurine transport in MDCK cells grown on porous supports and then studied the effect of hypertonicity which is known to elicit increased uptake of some other nonperturbing osmolytes by MDCK cells. Basal uptake exceeded apical uptake, with Km and Vmax of 56 microM and 933 pmol/min.mg protein on the basal surface and 10 microM and 50 pmol/min.mg protein on the apical surface. On both surfaces, virtually all taurine uptake was Na+ and Cl- dependent. 24 h after cells were shifted to hypertonic medium (500 mosmol/kg), taurine uptake doubled on the basolateral surface without change on the apical surface. The response to hypertonicity was the result of an increase in Vmax without change in Km. There was no change in taurine efflux when cells were shifted from isotonic to hypertonic medium. When cells adapted to hypertonic medium were shifted to isotonic medium, a large transient basolateral efflux of taurine occurred within 10 min. We conclude that taurine can function as a nonperturbing osmolyte in MDCK cells and that tonicity-regulated taurine transport is a basolateral function in MDCK cells.

Pulmonary Macrophages Can Stimulate Cell Growth of Bovine Bronchial Epithelial Cells

Macrophages are thought to participate in tissue repair following injury by releasing growth factors into the local environment. To evaluate whether pulmonary macrophages can mediate airway epithelial repair, we attempted to determine if pulmonary macrophages can stimulate growth of bovine bronchial epithelial cells in vitro. Bronchial epithelial cells isolated by protease digestion of the bovine bronchi were plated into tissue culture dishes with and without macrophage-conditioned medium. Bronchial epithelial cells cultured with macrophage-conditioned medium showed a significantly greater cell growth than those without macrophage-conditioned medium when assessed by direct enumeration of the cell numbers and by clonal growth assay. Stimulation of proliferation was confirmed by autoradiography using [3H]thymidine uptake into cell nuclei. Co-culture of pulmonary macrophages with bronchial epithelial cells also led to an increase in cell number. Immunohistochemical staining of the proliferating cells showed that these cells were positively stained by anti-keratin antibodies confirming that they were bronchial epithelial cells. Partial characterization of the activity in macrophage-conditioned medium showed that it was nondialyzable, pepsin- and acid-labile, and lipid-inextractable. Sephadex G-75 column fractionation indicated this activity existed in a high molecular fraction, thus suggesting a peptide. DEAE ion exchange chromatography revealed 3 peaks of stimulating activity. One peak resulted in a decrease in cell number, suggesting a possible inhibitory activity. The DEAE results thus suggest that macrophages may release several factors that can affect bronchial epithelial cell proliferation. In conclusion, pulmonary macrophages stimulate cell proliferation of bronchial epithelial cells in vitro. The stimulatory activity that may be heterogeneous appears to have the properties of a peptide.

Optimization of culture conditions for human corneal endothelial cells

Long-term cultivation of human corneal endothelial cells (HCEC) was optimized with respect to different components of the culture system: 25 different nutrient media, different sera, 6 mitogens and various substrates were tested in their ability to influence clonal growth and morphology of HCEC. F99, a 1:1 mixture of the two media M199 and Ham's F12, was the most effective basal medium in promoting clonal growth of HCEC. Among various sera, human serum and fetal bovine serum showed optimal growth promoting activities in combination with F99, whereas newborn bovine serum (NBS) was by far superior for the development of a typically corneal endothelial morphology. Crude fibroblast growth factor (FGF), or alternatively endothelial cell growth supplement, was absolutely essential for clonal growth of HCEC at low serum concentrations, for example 5% NBS. Formation of a monolayer with a morphology similar to corneal endothelium in vivo was observed only on culture dishes coated with basal membrane components such as collagen type IV, laminin, or fibronectin. The most pronounced effect on morphologic appearance was obtained by culturing the cells on the extracellular matrix (ECM) produced by bovine corneal endothelial cells. Moreover, ECM could substitute for crude FGF in clonal growth assays.

Clonal growth of human prostatic epithelial cells is stimulated by fibroblasts

The interaction of fibroblasts with adult human prostatic epithelial cells was studied in vitro. Stimulation of epithelial cell proliferation, measured by clonal growth assay, was demonstrated when prostatic epithelial cells were grown in coculture with fibroblasts. Epithelial growth in cocultures with fibroblasts was greater than could be obtained in isolated culture in an "optimized," serum-free medium previously described. Fibroblasts were able to compensate for the deletion of several growth factors from this "optimized" medium, including epidermal growth factor and insulin, but were notably unable to replace bovine pituitary extract. Epithelial growth stimulation identical to that achieved in coculture was produced by medium conditioned by fibroblasts. Similar results were obtained by using fibroblasts of adult human prostate origin, adult human skin, human fetal lung fibroblasts (IMR-90), and mouse 3T3 cells. No difference in response was demonstrated between prostatic epithelial cells derived from normal or malignant tissues. Fixed fibroblast monolayers and extracellular matrix prepared from fibroblast cultures failed to stimulate prostatic epithelial growth. These results suggest that a soluble growth factor is secreted by prostatic fibroblasts and other human fibroblasts of nonprostatic origin, as well as by embryonic mesenchymal cells from nonhuman species, which is capable of producing a marked proliferative response in vitro from adult human prostatic epithelium. This proliferative response could not be reproduced by the addition of a variety of known growth factors to prostatic epithelial cell cultures.

Altered production of the active oxygen species is involved in enhanced cytotoxic action of acylated derivatives of ascorbate to tumor cells

Our previous study shows that 6-O-acyl derivatives of L-ascorbic acid inhibits more markedly cell growth of mouse Ehrlich carcinoma than ascorbic acid. The present study shows that 6-O-palmitoyl ascorbic acid but not ascorbic acid prolongs the lifespan of mice into which tumors such as Meth A fibrosarcoma, MM46 mammary carcinoma, Ehrlich carcinoma and sarcoma 180 are implanted. The potentiated cytotoxicity of 6-O-palmitoyl ascorbic acid is not due to an increase in duration time of the cytotoxic action, because 6-O-palmitoyl ascorbic acid is gradually inactivated during contact with tumor cells and exhibits a similar action time curve to that of ascorbic acid as shown by clonal growth assay. Cytotoxicity of 6-O-palmitoyl ascorbic acid is markedly diminished by combined addition of catalase and superoxide dismutase (SOD), as shown by dye exclusion assay, whereas the cytotoxicity was slightly reduced by either enzyme alone but not by the specifically inactivated or heat-denatured enzymes. In contrast, cytotoxicity of ascorbic acid is abolished by catalyse but not SOD. Autooxidation of 6-O-palmitoyl ascorbic acid was not inhibited by catalase plus SOD. The results indicate that cytotoxicity of 6-O-palmitoyl ascorbic acid is attributed at least partly to both hydrogen peroxide (H2O2) and superoxide (O2-.) generated at the early stage. Cytotoxicity of 6-O-palmitoyl ascorbic acid is also appreciably attenuated by singlet oxygen (1O2) scavengers such as hydroquinone, 1,4-diazobicyclo-2,2,2-octane or sodium azide, but not by hydroxyl radical scavengers including butylated hydroxytoluene, D-mannitol, benzoic acid and ethanol. Thus, in contrast to cytotoxicity of ascorbic acid mediated entirely by H2O2 initially generated, acylated ascorbic acid produces a diversity of active oxygen species including H2O2, O2-. and other species secondarily generated via disproportion, which may be additively involved in the enhanced cytotoxic action.

Altered production of the active oxygen species is involved in enhanced cytotoxic action of acylated derivatives of ascorbate to tumor cells

Our previous study shows that 6-O-acyl derivatives of l-ascorbic acid inhibits more markedly cell growth of mouse ehrlich carcinoma than ascorbic acid. The present study shows that 6-O-palmitoyl ascorbic acid but not ascorbic acid prolongs the lifespan of mice into which tumors such as Meth A fibrosarcoma, MM46 mammary carcinoma, Ehrlich carcinoma and sarcoma 180 are implanted. The potenciated cytotoxicity of 6-O-palmitoyl ascorbic acid is not due to an increase in duration time of the cytotoxic action, because 6-O-palmitoyl ascorbic acid is gradually inactivated during contact with tumor cells and exhibits a similar action time curve to that of ascorbic acid as shown by clonal growth assay. Cytotoxicity of 6-O-palmitoyl ascorbic acid is markedly diminished by combined addition of catalase and superoxide dismutase (SOD), as shown by dye exclusion assay, whereas the cytotoxicity was slightly reduced by either enzyme alone but not by the specifically inactivated or heat-denatured enzymes. In contrast, cytotoxicity of ascorbic acid is abolished by catalyse but not SOD. Autooxidation of 6-O-palmitoyl ascorbic acid was not inhibited by catalase plus SOD. The results indicate that cytotoxicity of 6-O-palmitoyl ascorbic acid is attributed at least partly to both hydrogen peroxide (H2O2) and superoxide (O2⨪) generated at the early stage. Cytotoxicity of 6-O-palmitoyl ascorbic acid is also appreciably attenuated by singlet oxygen (1O2) scavengers such as hydroquinone, 1,4-diazobicyclo-2,2,2-octane or sodium azide, but not by hydroxyl radical scavengers including butylated hydroxytoluene, d-mannitol, benzoic acid and ethanol. Thus, in contrast to cytotoxicity of ascorbic acid mediated entirely by H2O2 initially generated, acylated ascorbic acid produces a diversity of active oxygen species including H2O2, O2⨪ and other species secondarily generated via disproportion, which may be additively involved in the enhanced cytotoxic action.

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using BALB/c 3T3 mouse embryo fibroblasts.

A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-D-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1:1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 micrograms/ml human transferrin, 100 micrograms/ml ovalbumin, and 1.0 microM dexamethasone. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-D-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of BFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50) ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50 of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in culture.

Growth factor involvement and oncogene expression in prostatic tumours

The effects of EGF, TGFα and 5α-dihydrotestosterone on the growth of a prostatic epithelial cell line have been evaluated in clonal growth assays. Similar bioassay systems have been used to identify tumour-associated growth promoters derived from a human prostatic carcinoma cell line (PC3). Growth factor activity was associated with proteins of M r 20–30 kDa. In a separate study, EGF receptor concentration and cellular proto-oncogene expression was assessed in prostatic tumour samples. In prostatic carcinoma samples, strong correlation was observed between EGF receptor concentration and c- myc expression. There were no significant correlations between EGF receptor concentration and tumour grade or androgen receptor content in carcinoma samples. EGF receptor concentration was significantly higher in prostatic carcinoma specimens than in BPH.

Response of Carcinogen-Altered Mouse Epidermal Cells to Phorbol Ester Tumor Promoters and Calcium

Primary cultures of mouse epidermal cells are induced to terminally differentiate when extracellular calcium levels are increased to more than 0.1 mM. After carcinogen treatment, cellular foci can be selected that resist this calcium signal to terminally differentiate. Calcium causes these foci to stratify; however, in contrast to normal epidermis, DNA-synthesizing cells in these foci are found in the suprabasal cell layers as well as in basal cells. Cell lines derived from these foci may be considered to be putative initiated cells. Three of these cell lines, designated 308, D, and F, have been characterized for their response to calcium and phorbol ester tumor promoters. The formation of cornified cells and the activity of epidermal transglutaminase were utilized as markers of epidermal differentiation. Neither calcium nor the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased transglutaminase activity or cornification of any of the 3 lines. Proliferation was estimated by the [3H]thymidine labeling index, by incorporation of [3H]thymidine into DNA, and by a clonal growth assay. Unlike primary normal cultures, raising the calcium level of the medium did not markedly reduce the rate of proliferation of any of the 3 cell lines. In 2 of the lines, line 308 and line D, proliferation increased in response to TPA exposure. In line F, [3H]thymidine incorporation in confluent cultures was inhibited by TPA, while in cells plated at clonal densities, TPA was cytotoxic at doses of 5 ng/ml or higher. If these calcium-resistant epidermal cell lines correspond to initiated cells, their lack of sensitivity to the induction of terminal differentiation by TPA could account for their growth relative to normal cells. Those lines that also respond to stimulation of proliferation by TPA to a greater extent than normal cells would have a further growth advantage.