Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex.

BACKGROUND: The protein kinase C (PKC) family has been implicated in the control of many cellular functions. Although PKC isotypes are characterized by their allosteric activation, phosphorylation also plays a key role in controlling activity. In classical PKC isotypes, one of the three critical sites is a carboxy-terminal hydrophobic site also conserved in other AGC kinase subfamily members. Although this site is crucial to the control of this class of enzymes, the upstream kinase(s) has not been identified. RESULTS: A membrane-associated kinase activity that phosphorylates the hydrophobic site in PKCalpha was detected. This activity was suppressed when cells were pretreated with the immunosuppresant drug rapamycin or the phosphoinositide (Pl) 3-kinase inhibitor LY294002. These pretreatments also blocked specifically the serum-induced phosphorylation of the hydrophobic site in PKCdelta in vivo. The most highly purified hydrophobic site kinase preparations ( approximately 10,000-fold) reacted with antibodies to PKCzeta/iota. Consistent with this, rapamycin and LY294002 reduced the recovery of PKCzeta from the membrane fraction of transfected cells. An activated mutant of PKCzeta, but not wild-type PKCzeta, induced phosphorylation of the PKCdelta hydrophobic site in a rapamycin-independent manner, whereas a kinase-dead PKCzeta mutant suppressed this serum-induced phosphorylation. The immunopurified, activated mutant of PKCzeta could phosphorylate the PKCdelta hydrophobic site in vitro, whereas wild-type PKCzeta could not. CONCLUSIONS: PKCzeta is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCdelta hydrophobic site in vitro and in vivo. PKCzeta can therefore control the phosphorylation of this PKCdelta site, antagonizing a rapamycin-sensitive pathway.

Comparative cell killing and kinetic effects of vincristine or vindesine in mammalian cell lines.

Survival curves for several monolayer and suspension cultures following 24-hour exposures to various vincristine (VCR) or vindesine (VDS) concentration were all of the exponential-plateau type. Cytotoxicity increased with duration of exposure in hamster NIL8 cells and was comparable for both drugs. Murine L5178Y cells, although 200 times more sensitive than NIL8 cells, also showed similar sensitivities to VCR and VDS. Murine neuroblastoma cells proved approximately fivefold less sensitive to VDS than VCR, whereas qualitative and quantitative differences in the activity of these two drugs were noted in human neuroblastoma cells. The lethal effects of VCR and VDS were exerted solely on logarithmically growing NIL8 cells in the S-phase. Plateau-phase human neuroblastoma cells were also significantly less sensitive to both drugs than were logarithmically growing populations, were cell kill occurred during the S-phase. Complete mitotic arrest, induced by a 24-hour exposure to VCR or VDS, was only partially reversible, whereas rapid and complete reversibility occurred after only 5 hours of drug exposure.

Overexpression of P-glycoprotein in mammalian tumor cell lines after fractionated X irradiation in vitro.

We observed that in vitro exposure of mammalian tumor cells to fractionated x irradiation results in the expression of drug resistance. The cause of this resistance was investigated in a series of Chinese hamster ovary cell lines that had survived exposure to multiple lethal doses of radiation. These cell lines had increased levels of P-glycoprotein (Pgp), the multidrug-resistance-associated membrane glycoprotein. Consistent with the classic multidrug resistance phenotype, they exhibited cross-resistance to multiple drugs, as well as sensitivity to reversal of vincristine resistance by verapamil. However, the cell lines showed no change in their sensitivity to x rays. Pgp overexpression occurred in these cells, despite a lack of Pgp gene amplification or of significant alteration in Pgp messenger RNA levels. Although the cause of increased Pgp levels is not yet known, these data suggest a biological basis for the clinical problem of drug resistance that can occur in previously irradiated tumors.

Cytotoxic effects and biological activity of 2-aza-8-germanspiro[4,5]-decane-2-propanamine-8,8-diethyl-N,N-dimethyl dichloride (NSC 192965; spirogermanium) in vitro.

Lethal and other biological effects of 2-aza-8-germanspiro[4,5]decane-2-propanamine-8,8-diethyl-N,N-dimethyl dichloride (NSC 192965; spirogermanium), representing a new chemical class of compound exhibiting antitumor activity, have been studied in vitro. Survival curves for NIL 8 hamster cells were exponential with greater kill occurring with increasing drug concentrations and longer exposure times. Cytotoxicity was temperature dependent. "Quiescent" cultures were significantly less sensitive to spirogermanium than were logarithmically growing cells. These lethal effects showed no phase specificity. There was no evidence of progression delay through the cycle following spirogermanium treatment. When spirogermanium was tested against a range of human cell lines, the consistency of the values for the drug concentration required to reduce survival by 50% on the exponential part of the survival curve, derived from colony-forming assays, was most marked. The survival curves, characterized by an initial shoulder, were steep and exponential with measurements possible over only a narrow concentration range since complete cell lysis occurred at levels causing a greater than 2-log kill. Cell membrane damage by spirogermanium, as judged by dye exclusion, was progressive with time and increasing drug concentrations. Protein synthesis proved most susceptible to the drug. Spirogermanium concentrations cytotoxic to tumor cells were also toxic to cultured rat neurons, confirming the clinical neurological toxicity encountered. The precise mode of action of spirogermanium remains to be established, and these data further illustrate its apparent lack of specificity.

Characterization of a cisplatin-resistant human ovarian carcinoma cell line expressing cross-resistance to 5-fluorouracil but collateral sensitivity to methotrexate.

In vitro exposure of the TR170 ovarian carcinoma cell line to six intermittent 24-h treatments with a 90% inhibitory concentration of cisplatin (CDDP) (0.15 micrograms/ml; 0.5 microM) resulted in a 2-fold stably resistant subline designated TR170/CP+ (B.T. Hill et al., Int. J. Cancer, 39: 219-225, 1987). Resistance to CDDP in these CP+ cells has now been associated with reduced uptake of 195mCDDP (2-fold; P less than 0.01) and decreased removal of specific Pt-DNA adducts, quantitated immunochemically, indicative of an apparent increased tolerance of CDDP-induced DNA damage. Specifically these resistant cells appeared deficient in removal of the major cis-Pt-(NH3)2d(pGpG) adduct and the difunctional cis-Pt(NH3)2d(GMP)2 lesion, showed less efficiency in removing cis-Pt(NH3)2d(pApG) adducts, but proved as proficient as the parental cell line in removing DNA-DNA interstrand cross-links. Activities of DNA polymerase-alpha and -beta were comparable in both lines, and no significant alterations in glutathione metabolism were identified. Response to acute X-irradiation was not modified in these TR170/CP+ cells, but they showed marked (10-fold) cross-resistance to 5-fluorouracil and, unusually, proved collaterally sensitive (12-fold) to methotrexate. Resistance to 5-fluorouracil was associated with significantly increased thymidylate synthase activity (P less than 0.01), but this was not reflected in altered gene expression, while increased sensitivity to methotrexate was accompanied by increased drug uptake but by unaltered activity and expression of dihydrofolate reductase. These results indicate that exposure to CDDP can result in numerous alterations, both intracellularly and at the cellular membrane, reflected in significant changes in the tumor cells' responses to the cytotoxic effects of a range of antitumor drugs. The clinical relevance of these observations remains to be established.

Loss of protein kinase C function induces an apoptotic response.

A correlation has been observed between the dephosphorylation and downregulation of cPKC and the induction of apoptosis. This relationship is shown to be causal in COS cells where expression of dominant negative PKC alpha is shown to induce apoptosis. This response is rescued by co-expression of wild-type PKC alpha. The results demonstrate that PKC provides a survival signal that can be supplied by PKC alpha alone.

Characterization of the P-glycoprotein over-expressing drug resistance phenotype exhibited by Chinese hamster ovary cells following their in-vitro exposure to fractionated X-irradiation.

Exposure of the Chinese hamster ovarian AuxB1 cell line in vitro to fractionated X-irradiation generated sublines designated DXR-10, which proved resistant to multiple drugs and overexpressed P-glycoprotein (Pgp), as judged by Western blotting using the C219 monoclonal antibody. Further characterization of these irradiated DXR-10 sublines has provided evidence for: (i) the expression of cross-resistance to gramacidin D, taxol, puromycin and Navelbine, but not to daunomycin or mitoxantrone; (ii) overexpression of the class I Pgp, as judged by Western blotting using the C494 monoclonal antibody; (iii) decreased accumulation of 3H-vincristine, which could be enhanced by verapamil addition; (iv) unaltered accumulation and subcellular distribution of adriamycin; (v) significantly increased rhodamine 123 accumulation in the presence of verapamil; (vi) plasma-membrane ultrastructural modifications resulting in a significantly increased surface area; (vii) numerous clonal karyotypic alterations, with abnormalities involving the long arm of chromosome 1 being consistently identified; (viii) a lack of overexpression of sorcin; (ix) increased total glutathione levels and overexpression of glutathione S-transferase pi. The fact that only certain of these features are considered characteristic of the 'classic' multidrug-resistant CHRC5 cell line supports our earlier proposal that exposure to fractionated X-irradiation results in the expression of a unique drug-resistance phenotype.

Differential expression of drug resistance following in vitro exposure of human tumour cell lines to fractionated X-irradiation.

We have established that drug resistance can be expressed following in vitro exposure of tumour cells not only to antitumor drugs but also to fractionated X-irradiation. These data therefore suggest a biological basis for the clinical problem of drug resistance that can occur in patients with previously irradiated tumors. These observations, if confirmed, have clinical implications for the combined modality approach and need to be considered when attempting to identify resistant tumour cells in clinical specimens with the aim of monitoring or identifying effective drug regimens.

Evidence that transcription changes in ageing cultures are terminal events occurring after the expression of a reduced replicative potential.

There is a progressive decline in replicative capacity with increasing age as expressed in terms of percentage labelled nuclei with 3H-thymidine and altered saturation density at confluency. This expression of ageing in vitro is seen in three different lines of human embryo diploid fibroblasts, although the pattern and rate of decline is different in each case. Generalization about in vitro ageing from studies with one cell line should therefore be made with care or avoided. There was an increase in total cellular RNA content as cultures aged which was more pronounced as cells entered the senescent or terminal phase of their lifespan. This increase appeared to be accompanied by a slightly elevated uptake and incorporation of 3H-uridine per cell. Template activity of isolated nuclei was markedly reduced in very late passage or phase III cells, but did not show a progressive decline with increasing age. These studies show that there is a reduced replicative potential which is not accompanied by a detectable decline in transcription, and suggest that the altered template activity should be regarded as an effect of ageing in vitro.

Studies of variation in inherent sensitivities to radiation, 5-fluorouracil and methotrexate in a series of human and murine tumor cell lines in vitro.

Clinical studies have reported reduced response rates to subsequent chemotherapy in certain tumors recurring after radiotherapy. We have investigated whether there are any correlations between radiation and drug responses in vitro using a range of murine and human tumor cell lines. We have compared sensitivities to X-irradiation and to 24 hr exposures to two widely used antitumor drugs, methotrexate and 5-fluorouracil. The 4 murine lines selected showed a range of radiation responses with Do values of 0.48-0.76 Gy. Methotrexate sensitivities also exhibited an 800-fold difference (IC50 values of 0.018-14.9 micrograms/ml) which appeared to correlate inversely with radiation response. Sensitivity to 5-FU was less variable in these cells with a range of IC50 values of 0.056-0.43 microgram/ml and was unrelated to radiation response. In contrast, in the human lines tested, no correlations were observed between drug sensitivities and radiation response. The six lines tested (3 derived from epidermoid head and neck tumors, 2 from neuroblastomas and 1 from a colon carcinoma) showed a range of radiation responses with Do values of 0.66-1.59 Gy. Methotrexate sensitivities ranged only over a 150-fold concentration but, contrasting with data from the murine cells, no correlation with radiation response was apparent. Similarly, no correlations between response to 5-fluorouracil and radiation or 5-fluorouracil and methotrexate were noted, which is inconsistent with results using murine cells.

An evaluation of the role of glutathione and its associated enzymes in the expression of differential sensitivities to antitumour agents shown by a range of human tumour cell lines.

Glutathione and its associated enzyme activities have been quantitated in a series of human tumour continuous cell lines expressing a range of in vitro sensitivities to certain antitumour agents. Fourteen different parental lines and 15 various drug- and X-ray-selected resistant sublines have been studied. Quantitative relationships between total glutathione levels and related enzyme activities and sensitivities to six clinically-useful antitumour drugs or X-rays, as judged by colony forming assays, have been determined by linear regression analysis. A positive correlation has been identified between glutathione levels and sensitivities to cisplatin. Adriamycin, or to X-rays. In addition, positive correlations were noted between cisplatin sensitivities and glutathione peroxidase and reductase activities and for Adriamycin responses with respect to glutathione peroxidase activity, using cumene hydroperoxide as substrate. However, no positive correlations were noted for glutathione levels or these enzyme activities with differential methotrexate, etoposide, vincristine or 5-fluorouracil cytotoxicities. Furthermore, no direct relationship was apparent between total glutathione S-transferase activities and any of these drug or X-ray sensitivities in this series of cell lines. These data appear to provide further evidence linking altered glutathione metabolism with differential cytotoxicities of certain clinically-useful antitumour agents.

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

A 27 kDa heat shock (HSP27) has been analysed 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 degrees 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.

Cytogenetic analysis of four human ovarian carcinoma cell lines.

Four cell lines derived from adenocarcinomas of the ovary, including three recently established cell lines, have been karyotyped. Chromosomes #1, #3, and #6 were found to be frequently involved in translocations with various other chromosomes, in agreement with results of other investigators, strongly implicating genes on these chromosomes in ovarian tumorigenesis.

Establishment of Vincristine-resistant and vindesine-resistant lines of murine lymphoblasts in vitro and characterisation of their patterns of cross-resistance and drug sensitivities.

Clinical evidence suggests some lack of cross resistance between vincristine (VCR) and vindesine (VDS). To investigate this phenomenon experimentally, drug-resistant L5178Y lymphoblast cell lines have been derived in vitro. These lines, under conditions of continuous drug exposure, exhibit a 50-fold order of resistance. Resistance appears due, at least in part, to impaired cellular drug accumulation and retention. Exposure of these resistant cells to VCR or VDS for 24 h showed that the presence or absence of cross resistance was dose-dependent, being most noticeable at low concentrations (less than 0.5 ng/ml) and absent at higher drug levels. Cross resistance also showed some dose-dependency for vinblastine and formyl-leurosine, but this was not seen with other drugs. Marked and complete cross resistance at all concentrations tested was noted with adriamycin and 4'-epiadriamycin in both resistant lines, which, however, retained the same sensitivities as the parent line to VM26, VP-16-213, 5-fluorouracil, and methotrexate. Responses to actinomycin D and mAMSA differed in these two resistant lines. VDS-resistant cells exhibited cross resistance to both drugs, whilst VCR-resistant cells showed only slight resistance to actinomycin D whilst retaining full sensitivity mAMSA. This observation that cross resistance between VCR and VDS is not invariable in vitro appears to reflect clinical experience.

Identification of anthracycline analogues with enhanced cytotoxicity and lack of cross-resistance to adriamycin using a series of mammalian cell lines in vitro.

Clinical resistance to adriamycin (ADR) develops readily, and cardiotoxicity is a major dose-limiting side effect. A range of anthracycline derivatives have been synthesized recently, and a number reported to exhibit significantly reduced cardiotoxicity in experimental animals. Using NIL 8 Syrian hamster overy cells and two continuous human tumour cell lines derived from colon carcinomas we have screened a series of 11 anthracycline analogues, determining their in vitro cytotoxic effects by colony-forming assays. Five agents proved significantly more cytotoxic than ADR: dihydroxyanthraquinone (DHAQ), mitoxantrone (DHAD), 4-demethoxydaunorubicin (4-DNR), 4'-0-tetrahydropyranyl-adriamycin (THP-ADR), and 4'-deoxyadriamycin (4-ADR). We have also established in vitro a subline of the L5178Y murine lymphoma resistant to ADR and have used this model to identify derivatives with potential value for overcoming ADR resistance. We have observed three patterns of response: (i) complete cross-resistance with 4'-epiadriamycin and daunorubicin; (ii) slight cross-resistance with 4-DNR, THP-ADR, 7-con-O-methyl-nogarol and aclacinomycin A; and (iii) complete absence of cross-resistance with 4-ADR, 4'-O-methyladriamycin, DHAQ, DHAD, and methylhydroxyellipticinium. These straightforward preclinical screens thus identify three drugs which may merit clinical evaluation, since they not only show an increased level of cytotoxicity in vitro to ADR at equivalent concentrations but also overcome resistance to ADR in this murine model system.

A comparative assessment of the in vitro effects of drugs on cells by means of colony assays or flow microfluorimetry.

The effects of a range of anticancer drugs on murine neuroblastoma cells, used as a readily reproducible model system, have been compared by means of colony-forming assays and analyses by flow microfluorimetry (FMF). FMF provides the most rapid means of assessing the kinetic effects of drugs on cells. However, interpretation of these data is not clear-cut, since drug effects are highly dose-dependent and no distinction can be made easily between progression arrest and cell kill. Thus whilst FMF allows some qualitative assessment of the perturbing effects of cytotoxic drugs quantitative evaluation of cytotoxicity is still dependent on data from the more time-consuming cloning assays. However, when cells are treated with certain drugs, e.g., methotrexate, vincristine, or VM26, for only 1 h, negligible kill occurred as measured by colony formation. Therefore it appears necessary to prolong in vitro exposure time when testing these drugs or evaluating cytotoxicity of potential antitumour agents in vitro.

Comparative effectiveness of mitoxantrone and doxorubicin in overcoming experimentally induced drug resistance in murine and human tumour cell lines in vitro.

Using a range of cell lines of murine and human tumour origin in which relatively modest levels (2- to 17-fold) of drug resistance have been selected in vitro by exposure to a range of standard antitumour drugs, we compared the cytotoxic effects of doxorubicin (DOX) and mitoxantrone (MITO). In general, significantly lower concentrations of MITO than of DOX were required to achieve comparable cytotoxicity, confirming previously published data. MITO appears more generally effective against the murine L5178Y drug-resistant sublines than DOX, although there was no expression of collateral sensitivity to this newer agent. In the various human tumour lines there was a lack of cross-resistance to both DOX and MITO in two 5-fluorouracil (FU)-resistant lines and one of two cisplatin (CDDP)-resistant cells, but cross-resistance was expressed in one subline resistant to vincristine (VCR) and two etoposide (VP-16)-resistant sublines. One murine and two human DOX-resistant sublines were effectively killed by MITO, whilst DOX proved effective against the human MITO-resistant subline. This apparent lack of cross-resistance between DOX and MITO in these resistant sublines expressing low levels of resistance in vitro therefore appears to contrast with previous reports involving highly multidrug-resistant DOX-selected sublines. However, since the latter lines generally exhibited profound cross-resistance to VCR and definite cross-resistance to VP-16, this may at least in part dictate their responses to MITO. Therefore, attempts to use experimentally derived drug-resistant sublines for preclinical drug screening should be approached with caution, since patterns of drug response appear to be influenced by the level of drug resistance expressed. The need remains to determine which type of model system provides the most relevant clinical information.

A comparison of the lethal and kinetic effects of doxorubicin and 4'-epi-doxorubicin in vitro.

"In vitro" lethal an kinetic effects of 4'-epi-doxorubicin (EPI-DXR) have been established and compared with those of doxorubicin (14-hydroxy-daunorubicin, adriamycin, NSC-123127, DXR). Both drugs show comparable cytotoxicity against a range of murine and human cell lines. Cytotoxicity increases exponentially with drug concentration and with duration of exposure. EPI-DXR like DXR exerts maximal lethal effects during the late S and G2 phases of the cycle in synchronised NIL8 Syrian hamster cells. Flow microfluorimetric data and measurements of mitotic indices provide evidence of population arrest in G2 with both drugs. Responses of various drug-resistant L5178Y cell lines were similar for DXR and EPI-DXR: (i) DXR-resistant cells exhibit complete cross-resistance to EPI-DXR, (ii) vincristine-resistant cells are cross-resistant to DXR and EPI-DXR, and (iii) methotrexate-resistant and 5-fluorouracil-resistant cells show collateral sensitivity to both drugs. These studies emphasise the similarities of DXR and EPI-DXR.