Insensitive Cell Lines Research Articles

Metabolism of N-[4-hydroxyphenyl]retinamide (4-HPR) to N-[4-methoxyphenyl]retinamide (4-MPR) may serve as a biomarker for its efficacy against human breast cancer and melanoma cells.

A clinical trial of N-[4-hydroxyphenyl]retinamide (4-HPR) has been in progress for the past 4 years to evaluate its role in chemoprevention of breast cancer. However, it is currently not known whether the effect of 4-HPR in breast cells is mediated by 4-HPR directly or through one of its metabolites. In this report, we investigated in vivo and in vitro effects of 4-HPR on three different breast carcinoma cells and two different melanoma cell lines. In vitro, the growth of all three breast carcinoma cell lines was inhibited by 4-HPR. Only one of two melanoma cell lines (UISO-Mel-1) showed growth inhibition to 4-HPR. The cell lines sensitive to 4-HPR in vitro also showed inhibition to 4-HPR in a xenograft model. Dietary 4-HPR (0.5 mmol/kg diet) reduced the growth of UISO-BCA-1 xenografts in female athymic mice, but had no effect on UISO-Mel-6 xenografts. Metabolism investigations of the 4-HPR-sensitive and insensitive cell lines indicated that N-[4-methoxyphenyl]retinamide (4-MPR), the major metabolite of 4-HPR, was detected only in cells sensitive to 4-HPR. Further in vitro studies with 4-MPR suggested that it is not an active metabolite of 4-HPR as it failed to inhibit growth of 4-HPR-resistant UISO-Mel-6 cells, and showed no dose-dependent inhibition of 4-HPR-sensitive breast carcinoma and melanoma cell lines. Our results in the present study indicate that, although 4-MPR is not an active metabolite of 4-HPR, detection of this metabolite in the malignant cells may serve as an indirect biomarker to predict response of cells to 4-HPR.

IN VITRO SELECTION WITH FUSARIC ACID : A NOVEL APPROACH TO BREED FOR FUSARIUM RESISTANCE IN GLADIOLUS

In vitro selection represents a feasible method to isolate variants with improved traits. Selection in vitro for disease resistance is facilitated when well characterized toxins are used as selective agents. However the number of metabolic compound with a known role in the development of the disease is limited. The prospective to use a specific fungal metabolite as selective agent were investigated. We report on the use of fusaric acid for the selection of Fusarium-resistant plants. Before performing the in vitro selection, the possible use of fusaric acid (FA) as selective agent was evaluated by applying two assays involving shoots and callus tissue of 10 genotypes. Callus tissue could reflect only partially the Fusarium, resistance level of the genotypes while shoots cultured in presence of the toxin could reflect the real resistance of the genotype. Fusaric acid insensitive cell lines were selected form cell suspension challenged with 0.10–0.14 mM FA. The insensitivity to this toxin was retained even after the selective agent was removed. Callus originated from the cell lines grew well on a medium supplemented with 0.5 mM FA, when inoculated with a suspension of conidia the mycelial growth was significantly inhibited. Plantlets regenerated were tested with a shoot assay and half of them showed a reduced sensitivity to the toxin. Results suggest that selection of Fusarium-resistant variants through in vitro selection is a possible breeding approach. The final evaluation of the Fusarium-resistance level of the regenerated plantlets will be performed once the corms are grown to maturity.

DPC4 (SMAD4) mediates transforming growth factor-beta1 (TGF-beta1) induced growth inhibition and transcriptional response in breast tumour cells.

A family of structurally related proteins homologous to the Drosophila mothers against dpp (MAD) gene product have been implicated in signal transduction by members of the TGF-beta superfamily. One of these MAD related proteins (DPC4) has been cloned as a candidate tumour suppressor in pancreas carcinomas, suggesting a role for DPC4 in growth regulation by TGF-beta related proteins. The involvement of DPC4 in TGF-beta1 induced growth inhibition and transcriptional response is demonstrated here, by the introduction of DPC4 in the TGF-beta and activin insensitive breast tumour cell line MDA-MB-468, from which the DPC4 gene is deleted. Transfection of DPC4 in this cell line restores both growth inhibition and the induction of a TGF-beta sensitive reporter construct (3TPlux) by TGF-beta1. In contrast, a DPC4 splice variant lacking amino acid residues 223-301 and cloned from another TGF-beta and activin resistant breast tumour cell line (MDA-MB-231), does not restore the induction of the 3TPlux reporter by TGF-beta1. We also show that in this latter cell line activin resistance is partly due to the absence of a functional activin type IB receptor. These results indicate that DPC4 is part of the TGF-beta signalling cascade and mediates TGF-beta induced growth inhibition. Together with the deletion of DPC4 from pancreas carcinomas these results suggest a role for DPC4 as a tumour suppressor.

Differential Secretion of Cathepsins B and L from Normal and Tumor Human Lung Cells Stimulated by 12(S)-Hydroxy-eicosatetraenoic Acid

Cathepsins B and L play roles in intracellular and extracellular proteolysis in normal and malignant processes. A directed extracellular proteolysis by regulated secretion could facilitate the process of invasion. We have therefore investigated the effect of the physiological signal mediator 12(S)-hydroxy-eicosatetraenoic acid on the release of cathepsins B and L in normal and malignant human lung cells. Quantitative determinations of cathepsin activities were done by flow cytometry and spectrofluorometry using synthetic dipeptidyl substrates coupled to fluorogens. Most interestingly, a difference in the secretion of cathepsins B and L was found: only release of active cathepsin B was detected. The effect was specific for 12(S)-hydroxy-eicosatetraenoic acid, 12(R)-hydroxy-eicosatetraenoic acid, and 5(S)-hydroxy-eicosatetraenoic acid were ineffective. The response was immediate but a substantial amount of nonreleasable activity remained cell bound. Alveolar macrophages, Wi-38 fibroblasts, and tumor cells derived from large cell carcinomas and adenocarcinomas were sensitive to 12(S)-hydroxy-eicosatetraenoic acid, but cells from undifferentiated squamous cell carcinomas were not. Sensitivity did not parallel malignancy but more likely the degree of differentiation of cells. The investigated tumor cell lines showed no detectable endogenous 12-lipoxygenase activity to synthesize 12(S)-hydroxy-eicosatetraenoic acid from arachidonate; therefore, we assume a paracrine mechanism for 12(S)-hydroxy-eicosatetraenoic acid action. Protein kinase Cα, a key enzyme involved in 12(S)-hydroxy-eicosatetraenoic acid-elicited responses, was expressed in all sensitive tumor cells, but insignificantly in a sensitive normal cell line and an insensitive tumor cell line. From our experiments we propose two separate intracellular pools of active cathepsin B: an unreleasable, lysosomal fraction and a fraction available for regulated secretion. Different processing and sorting mechanisms may be responsible for the generation of these cathepsin B-fractions in these pools.

Second messenger up-regulation of androgen receptor gene transcription is absent in androgen insensitive human prostatic carcinoma cell lines, PC-3 and DU-145

A theoretical pathway of transcriptional regulation of the androgen receptor (AR) gene is via a cAMP response element (CRE) present in its promoter region (−508 to −501). After 20 h of stimulation with 8-bromo-cAMP, AR mRNA was upregulated in LNCaP but not in either PC-3 or DU-145 cell lines. We have demonstrated that the level of CRE binding protein (CREB) was the same in all cell lines and that the putative AR-CRE forms specific and competable protein interactions with CREB. The ability to regulate AR gene transcription via the second messenger pathway is lost in the PC-3 and DU-145 cell lines. This may be an important primary mechanism of androgen insensitivity in prostate cancer.

DIFFERENTIAL CARBOHYDRATE EXPRESSION IN TUMORIGENIC VS NONTUMORIGENIC PROSTATE CELL-LINES

Death from prostate cancer is most frequently a result of metastatic disease. A key step in the process of metastasis is the attachment of circulatory tumor cells to target organ endothelium. This process is thought to be mediated by lectins, a class of cell surface proteins that bind two or more carbohydrate groups. Using fluorescent microscopy and fluorescein isothiocyanate (FITC) conjugated lectins, the presence of various carbohydrates was examined in the following tumorigenic and non-tumorigenic cell lines: rat prostate epithelial (EPYP-2, EPYP-1), rat prostate endothelial (YPEN-2, YPEN-1, YPEN-2PV), Dunning rat prostate cancer cell lines (MLL, ML, AT6.3, AT.1, AT2.1, GP9F3), and three tumorigenic human prostate cell lines (LNCaP, PC3, PC3 bone). The lectin Triticum vulgaris (WGA) was found to readily bind the carbohydrates N-acetylglucosamine and sialic acid on the plasma membrane of tumorigenic cell lines. Interestingly, WGA bound carbohydrates located to the nucleus and cytoplasm in non-tumorigenic cell lines, indicating that N-acetylglucosamine and sialic acid residues are preferentially expressed on the cell membrane of prostate cancer cells. Lectin staining patterns in cell lines of varying metastatic potential revealed no significant difference between highly metastatic vs. low metastatic cell lines. Observations revealed an absence of N-acetylgalactosamine and L-fucose in all rat Dunning, epithelial and endothelial cell lines as well as all human prostate cancer cell lines except for the androgen insensitive human prostate cancer cell line PC3, in which L-fucose residues were detected in the nucleus and on the plasma membrane. PC3 bone cells, which metastasized selectively to bone, demonstrated the presence of galactose residues whereas PC3 cells did not, suggesting that preference for target organ endothelium may be influenced by the expression of carbohydrate residues. These data indicate that differential carbohydrate expression may play an important role in prostate cancer biology.

Mitomycin C Sensitivity in Human Bladder Cancer Cells: Possible Role of Glutathione and Glutathione Transferase in Resistance

In this study, we have examined the relationship between sensitivity to mitomycin C (MMC) and glutathione (GSH) and glutathione transferase (GST) levels using a panel of three unrelated human bladder cancer cell lines, J82, HT-1197, and SCaBER. Cell lines HT-1197 and SCaBER were about 2- and 4.5-fold more resistant to MMC as compared to J82. Although the GSH level did not differ significantly in these cell lines, GST activity in HT-1197 and SCaBER cells were higher by about 2.3- and 6.0-fold, respectively, as compared to J82. Similar to GST activity, GST π content was highest in the most insensitive cell line and lowest in J82 cells. The cytotoxicity of MMC was increased significantly in these cells by a 1-h pretreatment with a nontoxic concentration of ethacrynic acid (EA), an inhibitor of GST activity. EA pretreatment resulted in a marked GSH depletion as well as GST activity inhibition in both of these cells. Although pretreatment of J82 and SCaBER cells with a nontoxic concentration of D,L-buthionine- S,R-sulfoximine (BSO) caused similar GSH depletion, the cytotoxicity of MMC was enhanced only in SCaBER cells. The differential effect of BSO on MMC cytotoxicity in these cell lines appeared to be due to the differences in the extent of GSH regeneration after removal of BSO. While a marked GSH regeneration occurred in J82 cells within 1 h after BSO removal, such an effect was not observed in SCaBER cells. Combined treatment of these cells with BSO and EA produced a greater potentiation of MMC cytotoxicity in both the cell lines when compared to BSO or EA treatment alone. We conclude that GSH/GST levels may affect the sensitivity of human bladder cancer cells to MMC.

Relationship of HSP27 and oestrogen receptor in hormone sensitive and insensitive cell lines

A 27 kDa heat shock (HSP27) has been analyzed by immunoassay and immunoblotting in oestradiol sensitive and insensitive cells. Oestradiol growth responsive MCF7 and T47D human breast cancer cells and growth unresponsive variants derived therefrom have unaltered levels of HSP27 as well as retaining their oestradiol receptor phenotype. MCF7 cells induced to become doxorubicin resistant in culture lose both HSP27 and oestradiol receptor. Thus, in these three pairs of cells, HSP27 content parallels oestradiol receptor (ER). Analysis of a range of ER positive and negative human cell lines supports the positive relationship between HSP27 and ER. This included six ER positive and two ER negative breast tumour lines, one ER positive and one ER negative endometrial tumour cell line and seven ER negative human lines from other sites. One ER negative osteosarcoma line (HTB96) had appreciable levels of HSP27 that were unaffected after stable transfunction with an ER cDNA. Heat shock increases HSP27 levels in some but not all cell lines tested, the effect being inversely proportional to the basal (37°C) content. In a mouse mammary tumour cell line, loss of androgen sensitivity was accompanied by loss of HSP27. Loss of HSP27 occurred in MCF7 cells made drug resistant to Novatrone, vincristine and etoposide as well as doxorubicin; no detectable change was seen in cells made resistant by 5 fluorouracil or X-irradiation. In ER positive ZR75 human breast tumour cells and in both ER negative and positive variants of the HTB96 human osteosarcoma line, the intracellular distribution of HSP27 was analysed. Over 96% of the HSP27 was in the cytosol fraction and the distribution was unaffected by incubation with oestradiol. HSP27 has been discussed in the literature under three different names p29, p24 and HSP27. The data presented in this paper are reviewed in the context of the previous data. It is concluded that there is a good but not absolute correlation between the presence of ER and high amounts of HSP27 but that low amounts of HSP27 are present in many ER negative cells. The correlations between HSP27 and drug resistance are more complex.

Differential sensitivity of human myeloma cell lines and normal bone marrow colony forming cells to a recombinant diphtheria toxin-interleukin 6 fusion protein.

The cytotoxicity of a recombinant interleukin 6 (IL-6)-diphtheria toxin (DT) fusion protein towards human myeloma cell lines was investigated. DAB389-IL-6 inhibited protein synthesis and methylcellulose colony formation by U266 myeloma cells. In the clonogenic assay, the fusion protein approached the level of cytotoxicity achieved by native DT. The specificity of killing by DAB389-IL-6 was demonstrated by inhibition of cytotoxicity by a molar excess of free rhIL-6. The effect of DAB389-IL-6 on colony formation by six OCI-My cell lines was assessed. Similar to U266 cells, colony growth by the OCI-My 5 and -My 2 cell lines was inhibited in a simple dose dependent manner. However, a biphasic effect was observed for the IL-6 dependent OCI-My 4 cells; DAB389-IL-6 stimulated colony formation at low (< or = 10(-11) M) concentrations, yet was inhibitory at higher doses. Three other cell lines whose growth was not altered by IL-6 were relatively unaffected by DAB389-IL-6, despite their sensitivity to native DT. Flow cytometric analysis for IL-6 receptor expression using phycoerythrin-conjugated IL-6 demonstrated specific binding sites on both DAB389-IL-6 sensitive and certain insensitive cell lines, suggesting that other factors in addition to the expression of IL-6 receptors are involved in killing by the fusion toxin. Despite evidence for a role of IL-6 in myeloid cell development, normal bone marrow was insensitive to the IL-6 fusion toxin. In cultures containing both normal bone marrow and U266 cells DAB389-IL-6 effectively inhibited the growth of U266 myeloma colonies but had little effect on normal bone marrow erythroid, granulocyte and mixed erythroid/granulocyte colony growth. From these experiments we conclude that DAB389-IL-6 is specifically cytotoxic towards a subset of IL-6-responsive human myeloma cell lines and may be useful, in some cases, in the selective elimination of tumour cells from mixed populations of normal and malignant cells.

Valproic acid activity in androgen-sensitive and -insensitive human prostate cancer cells

Histone deacetylase (HDAC) inhibitors are currently undergoing clinical trials in cancer patients on the basis of their effect on proliferation, differentiation and apoptosis demonstrated in vitro. The goal of this study was to assess the effects of an HDAC inhibitor, valproic acid (VA), on proliferation, androgen-sensitivity, androgen receptor levels and E-cadherin (E-cad) expression in human prostate cancer cells. The effects of VA were evaluated in androgen-sensitive, LNCaP and -insensitive PC-3 human prostate cancer cell lines. Proliferation was assayed by cell counts and protein expression by Western blot analysis. Morphological changes were analysed under an inverted phase contrast microscope. High VA concentrations (1-25 mM) induced a very strong reduction in cell numbers ( approximately 90% with respect to control) of the two cell lines due to drug cytotoxicity. A low concentration (0.45 mM VA) slightly reduced (14%) LNCaP cell proliferation and abolished the response to androgen. In the PC-3 cells, the same concentration of VA had a more pronounced (40%) inhibitory effect and induced a response to dihydrotestosterone in terms of an enhancement in cell growth. These events were associated with morphological changes, an absence of cytotoxicity, an increase in androgen receptor levels, and, in PC-3 cells, an enhancement in E-cad expression which may be ascribed to VA differentiative action. Our findings, obtained with a VA dose (0.45 mM), which is consistent with plasma concentrations reached under oral administration of therapeutical doses in patients treated for different diseases, suggest that VA might have clinical value in prostate cancer therapy in androgen-sensitive and -insensitive tumors.

Antitumour synergism between non-toxic dietary combinations of isotretinoin and glucarate

Dietary calcium glucarate (CGT) increased the activity of non-toxic levels of dietary isotretinoin against pre-established tumors in the chemically-induced rat mammary tumour model. In the range of 1.0–1.5 mmol/kg diet, isotretinoin enhanced tumour growth by 20% over a 4 week course of treatment. Tumour growth inhibition not exceeding 15% was observed only at dosages as high as 2.0 mmol/kg, i.e. in the cumulative toxicity range. Growth inhibition by 64 mmol/kg diet of CGT alone was marginal, varying from zero to 8%. In contrast, the combination of 1.0 mmol/kg of isotretinoin and 64 mmol/kg of CGT caused a reversible inhibition of tumour growth, culminating in a net decrease in tumour volume of 20%. This study documents the marginal enhancement of tumour growth by high sub-optimal concentrations of isotretinoin alone, and describes conditions for inhibition of tumour growth by sub-optimal concentrations of the natural retinoid. Related in vitro studies on retinoid sensitive and insensitive cell lines suggest that the anticancer activity of the combination is dependent on sensitivity of the cells to retinoids.

Growth inhibition of Candida albicans by interleukin-2-activated splenocytes.

Murine splenocytes, Percoll-enriched low-density lymphocytes, and interleukin-2 (IL-2)-activated lymphocytes were assessed for the capacity to limit the growth of the hyphal form of Candida albicans. No fungal-growth-inhibitory activity was exhibited for C. albicans by either splenocytes or Percoll-enriched lymphocytes. These cells were capable of cytotoxic activity for a natural killer cell-sensitive cell line. However, when cultured for several days with IL-2, splenocytes acquired the capacity to inhibit the growth of the fungus. The appearance of the antifungal activity coincided with the development of cytotoxic activity for the natural killer cell-insensitive cell line. Anti-C. albicans and antitumor activities of IL-2-activated lymphocytes were competitively and reciprocally inhibited by C. albicans and the natural killer cell-sensitive and -insensitive cell lines. The antifungal activity of the IL-2-activated lymphocytes was exhibited against a number of clinical isolates of C. albicans and related fungal species. IL-2-activated human peripheral blood lymphocytes also acquired the capacity to inhibit the growth of C. albicans. These data show that in vitro growth inhibition can be mediated by IL-2-stimulated lymphocytes which are neither fungal strain nor mammalian species restricted in their biological activity.

Divergent responses to epidermal growth factor in hormone sensitive and insensitive human prostate cancer cell lines

The present study was undertaken to compare the relationship between response to exogenous epidermal growth factor (EGF) and the expression of the EGF-receptor (EGF-R) in an androgen sensitive (LNCaP) and insensitive (DU145) prostate cancer cell line. Although both cell lines demonstrated a single EGF-R binding site of similar high affinities (mean dissociation constant (Kd) +/- S.D. for DU145 = 1.0 +/- 0.6 nmol l-1; LNCaP = 2.8 +/- 2.2 nmol l-1) the number of binding sites (RT) for the hormone insensitive DU145 cells (mean +/- S.D. = 2.5 +/- 1.0 x 10(5) sites/cell) and 10-fold greater than that expressed in the androgen responsive LNCaP cell line (mean +/- S.D. = 2.0 +/- 1 x 10(4) sites/cell). Additionally exogenous EGF only minimally affected the growth and DNA synthesis of DU145 cells whereas LNCaP cells showed a significant response which was dose dependent. The autologous production of EGF-like molecules by DU145 cells is believed to reduce the cells needs for exogenous mitogens, thereby rendering the cells autostimulatory. Treatment of LNCaP cells with Mibolerone--a synthetic androgen--did not affect either the expression of the EGF receptor or the proliferative response observed with EGF. Western blot analysis, using monoclonal antibodies directed against the EGF receptor revealed a band of approximately 170 kD with DU145 cell lysates but the LNCaP EGF receptor was not detected using this technique.

Toxicity of the mycotoxins fumonisins B1 and B2 and Alternaria alternata f. sp. lycopersici toxin (AAL) in cultured mammalian cells

Fumonisins B1 and B2 and AAL toxin are a series of structurally related mycotoxins. Fumonisins B1 and B2, produced by Fusarium moniliforme Sheldon induce toxic hepatitis and hepatomas in rats and leukoencephalomalacia in horses. The cancer-promotion assay which has been used to guide their purification is slow and consumes large amounts of sample. We have examined a series of cultured mammalian cell lines in order to develop a more rapid and sensitive bioassay system, which may be useful for examining structure-activity relationships and the mechanism(s) of action of these toxins. Of 9 rat hepatoma cell lines tested, all except the two most de-differentiated line were sensitive to the three toxins, with a toxic response visible by 48 h. Approximate IC50 values for the most sensitive hepatoma line, H4TG, were 4, 2 and 10 micrograms/ml for fumonisins B1, B2 and AAL toxin, respectively, in 100 microliters cultures. Among 15 cell lines from other sources, only MDCK dog kidney epithelial cells were sensitive (IC50 = 2.5, 2 and 5 micrograms/ml, respectively). Studies in co-cultures of sensitive and insensitive cell lines and in cultures of a sensitive cell line over a range of cell densities indicated that cytotoxicity of fumonisins B1 and B2 does not involve metabolite activation to a derivative stable enough to diffuse to adjacent cells.

Production and response of a human prostatic cancer line to transforming growth factor-like molecules

Serum-free media conditioned by the androgen insensitive human prostate cancer cell line DU145 showed immunological transforming growth factor-alpha (TGF alpha) activity, as well as competing activity in epidermal growth factor (EGF) radioreceptor assays (RRA). Furthermore, there were factors in the conditioned media which inhibited and stimulated DNA synthesis by DU145 cells in a dose-dependent fashion. Fractionation of the concentrated conditioned media by reverse-phase high performance liquid chromatography revealed several peaks containing EGF-like competitive activity only one of which demonstrated TGF alpha activity. However, none of the peaks corresponded to immunoreactive EGF. Measurement of EGF receptors on DU145 cells by competition and saturation analysis revealed high levels of receptors (mean +/- s.d. = 2.5 +/- 1 x 10(5) surface receptors per cell) which were of high affinity (Kd +/- s.d. = 1.0 +/- 0.5 nmol l-1). Although DU145 cells express high levels of EGF receptors, DNA synthesis was only minimally affected by exogenous EGF and TGF alpha.

Die Rolle der Interferone beim Neuroblastom - Teil 1: Antiproliferative Effekte

Antiproliferative effects of interferon alpha, beta and gamma were investigated on several human neuroblastoma cell lines using the soft agar colony forming assay and the MTT-test. Investigations were carried out in order to prove whether there is any relationship between antiproliferative effects, inhibition of N-myc expression and the 2-5A system. Growth of neuroblastoma cells was inhibited by all three kinds of interferons in a concentration-dependent manner, however, rather high concentrations were necessary in some cell lines. Expression of N-myc oncogen was not inhibited by interferon-beta and no relationship between antiproliferative effects and the 2-5A system was observed. A vector containing a small N-myc fragment in antisense direction was constructed and transferred into the interferon insensitive human neuroblastoma cell line LS. After transformation, LS cells became sensitive to interferon beta: Proliferation as well as N-myc expression were inhibited and these processes are most probably associated with activation of the 2-5A system.

Target-Effector-Cell Interactions in the Human Natural-Killer(NK)-System: Isolation of Target Structures

The existence of structures on NK-sensitive target cells selectively recognized by the effector cells have been postulated. To test this hypothesis, four selected human cell lines were investigated for target-cell proteins which could serve as specific ligands for the putative NK-cell receptor(s). NP-40 extracts from two highly NK-sensitive (K 562 and Molt-4) and two rather insensitive cell lines (HL-60 and Reh-6) were fractionated on SDS-polyacrylamide gels and tested for their ability to inhibit binding of effector to target cells as well as NK cytotoxicity. Three fractions with molecular weights (MW) of 200, 120 and 80 ± 10 KD isolated from K 562 cells were able to inhibit binding of large granular lymphocytes (LGL) to K 562. Of the other cell lines, Molt-4 and HL-60, both which were able to inhibit lysis of K 562 in a cold target inhibition assay, showed also two inhibitory fractions with MW 120 and 80 KD, whereas Reh-6, which is not able to compete with 51Cr-labelled K 562 in a cytotoxicity assay, lacked these structures. The 200, 120 and 80 KD fractions isolated from K 562 and the 120 and 80 KID fractions from Molt-4 and HL-60 were able to inhibit lysis of K 562 cells when added to the cytotoxicity assay. By adsorption/elution of radiolabelled K 562 extracts to/from LGL it was possible to detect an 80 KID target-cell surface protein which became preferentially bound by LGL-enriched but not by LGL-depleted lymphocyte preparations. Our results indicate the existence of target-cell proteins in NK-sensitive cell lines which serve as specific ligands for binding of NK cells. These target-cell structures of human cell lines differed from NK target structures described for mouse-NK-sensitive cell lines.

Human natural killing against ovarian carcinoma.

Natural killing (NK) by lymphocytes from normal individuals against primary and established ovarian carcinoma cell lines was tested in short-term chromium release assays. Two established cell lines and 5/6 primary cell lines tested showed significant susceptibility to NK. Primary cell lines are, in general, less sensitive to NK than long-term cultured target cells. A common NK recognition determinant on ovarian carcinoma cells and on the erythroleukaemic K562 cells was demonstrated by cold target inhibition assays. The recognition structure was also present on an ovarian cell line resistant to NK but not on the insensitive leukaemic cell line, SB. The activity against ovarian carcinoma cells was associated with the presence of large granular lymphocytes (LGL) previously shown to be the major effector cells against K562 targets. In fractions obtained by Percoll gradient centrifugation of lymphocytes, only fractions with a high content of LGL demonstrated good NK activity. LGL mediated NK against both non-adherent K562 and the adherent ovarian carcinoma target cells independent of monocytes.

Mode of cell death induced in human lymphoid cells by high and low doses of glucocorticoid

The kinetics, specificity and morphology of cytolethal responses have been studied in human glucocorticoid-sensitive and -insensitive lymphoid cell lines (HLCL) and fibroblasts following treatment with high (10(-3)M) and low (10(-6)M) doses of steroid. The high dose cytolethal response appears non-specific occurring in all cell lines with every steroid tested. By contrast, the low dose (pharmacological) cytolethal response requires an active glucocorticoid and a sensitive HLCL. However, both high and low concentrations of steroid induce virtually identical morphological changes in dying cells and similar changes can be induced in cells killed by deliberate feed exhaustion. Although the morphological features in each case resemble apoptosis, the "programmed" physiological form of cell death, the intracellular events leading to cytolysis seem likely to differ. The earliest morphological changes presaging cell death comprise rounding up of cells and condensation of nuclear chromatin. Nuclear changes progress rapidly thereafter and appear to result from detachment of chromatin from the nuclear matrix. The low dose cytolethal response requires the continuous presence of glucocorticoid for periods in excess of 24h, prior to which cell growth appears unaffected. The constancy of this latent interval suggests glucocorticoids may influence some replication control mechanism unrelated initially to macromolecular biosynthesis.

Effects of deoxyribonuclease I and micrococcal nuclease on the release of dexamethasone-receptor complex from nuclei of sensitive and insensitive hepatoma cell lines

1. (1) Fu5 cells were sensitive to the glucocorticoid inhibition of cell growth and the hormonal induction of tyrosine aminotransferase (but not fructose-1, 6-bisphosphatase and glycogen synthase). AH-130 and AH-7974 cells were insensitive to both effects. 2. (2) The release of [ 3H]dexamethasone radioactivity from the nuclei of Fu5 and AH-130 cell preincubated with [ 3H]dexamethasone increased as the KCl concentration increased from 0 to 0.4 M, with no significant difference between the two cell lines. 3. (3) The radioactivity was more sensitively released in Fu5 nuclei than in AH-130 nuclie upon treatment with DNAase I. The release of radioactivity was always larger than the release of DNA in both cell nuclei. In contrast to DNAase I, micrococcal nuclease treatment did not show any difference between the two cell lines in the release of radioactivity from nuclei, always showing a release of radioactivity equal to that of DNA.