LY2 Cells Research Articles

P4-01-06: Global Characterization of the SRC-1 Transcriptome Identifies Disintegrin C as an ER-Independent Mediator of Endocrine Resistant Breast Cancer.

Abstract Background: The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity leading to the development of a steroid independent tumour. The p160 steroid coactivataor protein SRC-1 drives this tumour adaptability. SRC-1 is central to the development of the endocrine resistant phenotype and is an independent predictor of disease free survival in tamoxifen-treated patients. Here, using discovery studies we identify Disintegrin C (DC), a multidomain transmembrane glycoprotein as a direct, ER-independent target of SRC-1. Methods: SRC-1-ChIP sequencing was performed in endocrine resistant (LY2) cells. Microarray experiments were performed on LY2 cells transfected with scrambled siRNA or SRC-1 siRNA, +/−4-OHT to assess SRC-1-dependent gene expression. Combining the ChIP-seq and expression array datasets, a total of 2,065 genes were significantly downregulated (p<0.05) following SRC-1 knockdown. DC was selected for validation as a potential novel metastatic oncogene. DC transcipt levels were measured following knockdown and over-expression of SRC-1 and ERα. Mouse xenograft and SRC-1 knockout models were used to examine DC expression in vivo. A TMA comprising 560 patients was stained for DC. Functional validation studies were performed using 3D culture and migration assays, with transient knockdown of DC and treatment with JC1, an inhibitory peptide against DC. Results: Treatment with 4-OHT significantly increased the number of ChIP-enriched intervals identified. The majority of peaks in the 4-OHT treated sample were close to the transcriptional start site, in the promoter, first exon or upstream of the promoter. As a defined ER coactivator, an overlap between ER binding sites and those identified for SRC-1 is expected. 43% of high confidence SRC-1 peaks in 4-OHT-treated LY2 cells contained an ERE binding motif. This raises the possibility that, in endocrine resistance, SRC-1 can drive transcription independent of ER. Treatment with 4-OHT resulted in substantial recruitment of SRC-1 to the DC promoter. In primary tumours there was a significant association between transcript levels of SRC-1 and DC. Cells derived from mammary tumours of the SRC-1-/−/PyMT mouse lacked DC, but there was high expression in the wild type PyMT mouse. Knockdown of ERα in LY2 cells did not alter DC expression. Knockdown of DC reduced cell migration and restored cell differentiation in resistant cells. In xenograft studies treatment with 4-OHT increased tumour volume in the resistant tumours and induced DC expression. Treatment with recombinant JC1 reduced cellular migration in endocrine resistant cells. Kaplan Meier estimates of disease free survival show that DC is a strong predictor of disease free survival in breast cancer (p<0.0001). SRC-1 and DC are significant independent predictors of disease recurrence (odds ratios 2.18 and 2.4). Conclusion: Our discovery studies have uncovered a steroid independent SRC-1 mediated network in endocrine resistant breast cancer. We identified a new SRC-1 target, DC. DC represents a rational new therapeutic target with a robust companion biomarker for treatment of endocrine resistant tumours. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-06.

Differential expression of microRNA expression in tamoxifen-sensitive MCF-7 versus tamoxifen-resistant LY2 human breast cancer cells

Microarrays identified miRNAs differentially expressed and 4-hydroxytamoxifen (4-OHT) regulated in MCF-7 endocrine-sensitive versus resistant LY2 human breast cancer cells. 97 miRNAs were differentially expressed in MCF-7 versus LY2 cells. Opposite expression of miRs-10a, 21, 22, 29a, 93, 125b, 181, 200a, 200b, 200c, 205, and 222 was confirmed. Bioinformatic analyses to impute the biological significance of these miRNAs identified 36 predicted gene targets from those regulated by 4-OHT in MCF-7 cells. Agreement in the direction of anticipated regulation was detected for 12 putative targets. These miRNAs with opposite expression between the two cell lines may be involved in endocrine resistance.

Abstract 4329: In vitro and in vivo antitumor effects of gemcitabine in a murine head and neck squamous cell carcinoma (HNSCC) model

Abstract Background: Gemcitabine (2’2’-difluorodeoxycytidine/Gem) is a nucleoside analog and exerts its cytotoxicity by incorporating into the DNA during replication which leads to termination of chain elongation and tumor cell apoptosis. Chemotherapy with Gem is the standard of care for patients with pancreatic cancer and it has shown clinical efficacy and favorable toxicity profile in patients with advanced HNSCC. Goal: We evaluated the molecular aspects of Gem's anti-tumor effect in a highly metastatic orthotropic model of HNSCC, generated using a murine SCC cells (LY2) in BALB/c mice. Methods: Exponentially growing LY2 cells were treated with Gem (100nM-1µM) and its effect on cell survival, proliferation, apoptosis and signaling pathways involved cell growth were examined using MTT assay, Western blotting and immunohistochemical (IHC) assays for cleaved caspase 3 and proliferating cell nuclear antigen (PCNA). LY2 cells were intraorally implanted in BALB/c mice, after one week, tumor bearing mice were randomly divided into experimental (n=8) and control (n=8) groups and treated with intraperitoneal injections of Gem (100mg/Kg, twice a week for 3-weeks) and saline, respectively. Primary tumor growth rates were monitored weekly and tumor metastases were analyzed by histological examination. Results: LY2 cells were susceptible to killing by Gem (IC50 = 500 nM). The extracellular signal-regulated kinase (ERK) activation in LY2 cells was blocked immediately after the treatment with Gem. The activation of apoptosis as measured by the expression levels cleaved caspase 3, reached the highest level at 24 hours after the treatment. Gem treatment down-regulated the expression checkpoint kinase (CHK1) that protects cells from DNA damage induced cell death in LY2 cells. The mean apoptosis rate (% of cleaved caspase 3 positive cells detected by IHC) of LY2 cells treated with Gem was significantly higher (21%) compared to untreated control cells (< 5%). Interestingly, LY2 cells treated with Gem revealed significantly higher PCNA positive cells (76%) than untreated control cells (28%). Gem treatment of tumor bearing mice resulted in significant reduction tumor growth rate and incidence of regional lymph node metastases compared to the control group. Conclusion: The increase in PCNA positive cells within the LY2 cells treated with Gem compared to untreated control is counterintuitive to the anti-proliferative effect of Gem. Increased PCNA positive fraction of cells in Gem treatment group is most likely caused by S-phase arrest which has been reported to occur in cancer cells treated with Gem. Hence, PCNA cannot be used to determine the cell proliferation rates of tumor cells treated with Gem. Our data suggest that Gem has multiple innovative modes of anti-tumor activity against HNSCC which include DNA replication defect, loss of ERK activation and CHK1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4329. doi:10.1158/1538-7445.AM2011-4329

Abstract LB-148: XBP1 regulates NFkappaB signaling in antiestrogen resistant breast cancer cells

Abstract Unfolded protein response (UPR), a stress-induced survival mechanism, may be hijacked by cancer cells to avoid cell death. Therefore, the functional role of UPR during drug resistance has attracted an increasing amount of attention recently. Antiestrogen therapy, used in the treatment of estrogen receptor-positive (ER+) breast cancer, induces UPR. Upon endoplasmic reticulum stress, three arms of UPR signaling become activated to cope with stress conditions. One critical player that is regulated by two arms of the UPR signaling is a transcriptional factor called X-box binding protein 1 (XBP1). XBP1 exists in two forms, the transcriptionally inactive unspliced XBP1(U) and the spliced, actived XBP1(S). We have previously shown that overexpression of XBP1(S) confers estrogen independence and antiestrogen resistance in ER+ breast cancer cells. Others subsequently reported that XBP1(S) expression in ER+ breast tumors correlates with poor clinical responsiveness to Tamoxifen. However, the underlying signaling mechanisms affected by XBP1(S) as well as the effects of splicing on antiestrogen resistance remain unclear. We hypothesize that XBP1(S) mediates antiestrogen resistance in part through regulating nuclear factor kappa B (NFkappaB) signaling, since we previously showed that NFkappaB expression and activity are up-regulated in antiestrogen resistant cells, possibly through up-regulation of IKKgamma (NEMO) and RelA (NFkappaB p65). We now show that XBP1 regulates the expression of RelA in breast cancer cells. Overexpression of XBP1 in MCF7 and LCC1 antiestrogen sensitive breast cancer cells results primarily in an increase in XBP1(S) and an induction of RelA expression at both the mRNA and protein levels. We also show that NFkappaB transcriptional activity is upregulated by XBP1 overexpression. Moreover, the antiestrogen resistance conferred by XBP1 overexpression in MCF7 cells requires activated NFkappaB signaling. The presence of small molecule NFkappaB inhibitor, Parthenolide, sensitizes XBP1 ovexpressing MCF7 cells to Tamoxifen. Depletion of endogenous XBP1 using siRNA in antiestrogen resistant LCC9, LY2, and MCF7-RR breast cancer cells decreases RelA expression and NFkappaB transcriptional activity. Taken together, our findings establish a regulatory link between the UPR/XBP1 pathway and pro-survival NFkappaB signaling. Currently, we are actively investigating the role of XBP1 splicing in regulating NFkappaB signaling and antiestrogen resistance with a non-splicible mutant of XBP1 (XBP1(nonS)). In future studies, we will identify the pathways induced by XBP1/NFkappaB signals and further delineate their role in antiestrogen resistance in ER+ breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-148. doi:10.1158/1538-7445.AM2011-LB-148

Hypoxia-induced autophagic response is associated with aggressive phenotype and elevated incidence of metastasis in orthotopic immunocompetent murine models of head and neck squamous cell carcinomas (HNSCC)

Hypoxia confers resistance to chemoradiation therapy and promotes metastasis in head and neck squamous cell carcinomas (HNSCC). We investigated the effects of hypoxia in tumor phenotype using immunocompetent murine HNSCC models. Balb/c mice were injected intraorally with murine squamous cell carcinoma cells LY-2 and B4B8. Intratumoral hypoxia fraction was evaluated by the immunohistochemical detection of hypoxic probe pimonidazole and carbonic anhydrase IX (CAIX). Tumor cell apoptosis and autophagy in hypoxic areas of these tumors were examined immunohistochemically. Hypoxia-induced apoptotic and autophagic responses in vitro were examined by treating LY2 cells with CoCl2. B4B8 tumors exhibited a non-aggressive phenotype characterized by its slow growth rate and the lack of metastatic spread. LY2 tumors demonstrated an aggressive phenotype characterized by rapid growth rate with regional and distant metastasis. Intratumoral hypoxia fraction in B4B8 tumors was significantly lower than in LY2 tumors. The hypoxic areas in B4B8 tumors exhibited increased apoptosis rate than that of LY2 tumors. In contrast, the hypoxic areas in LY2 tumors revealed autophagy. The induction of hypoxia in vitro elicited autophagy and not apoptosis in LY2 cells. The induction of autophagy coupled with blockage of apoptosis in hypoxic areas promotes tumor cell survival and confers aggressive phenotype in immunocompetent murine HNSCC models.

IFNγ Restores Breast Cancer Sensitivity to Fulvestrant by Regulating STAT1, IFN Regulatory Factor 1, NF-κB, BCL2 Family Members, and Signaling to Caspase-Dependent Apoptosis

Antiestrogens are effective therapies for the management of many estrogen receptor-alpha (ER)-positive breast cancers. Nonetheless, both de novo and acquired resistance occur and remain major problems in the clinical setting. IFNgamma is an inflammatory cytokine that induces the expression and function of IFN regulatory factor 1 (IRF1), a tumor suppressor gene that can increase antiestrogen responsiveness. We show that IFNgamma, but not IFNalpha, IFNbeta, or fulvestrant (ICI; ICI 182,780; Faslodex), induces IRF1 expression in antiestrogen-resistant MCF7/LCC9 and LY2 cells. Moreover, IFNgamma restores the responsiveness of these cells to fulvestrant. Increased IRF1 activation suppresses NF-kappaB p65 (RELA) activity, inhibits the expression of prosurvival (BCL2, BCL-W), and induces the expression of proapoptotic members (BAK, mitochondrial BAX) of the BCL2 family. This molecular signaling is associated with the activation of signal transducer and activator of transcription 1 and leads to increased mitochondrial membrane permeability; activation of caspase-7 (CASP7), CASP8, and CASP9; and induction of apoptosis but not autophagy. Whereas antiestrogen-resistant cells are capable of inducing autophagy through IFN-mediated signaling, their ability to do so through antiestrogen-regulated signaling is lost. The abilities of IFNgamma to activate CASP8, induce apoptosis, and restore antiestrogen sensitivity are prevented by siRNA targeting IRF1, whereas transient overexpression of IRF1 mimics the effects of IFNgamma treatment. These observations support the exploration of clinical trials combining antiestrogens and compounds that can induce IRF1, such as IFNgamma, for the treatment of some ER-positive breast cancers.

Estrogen receptor alpha 46 is reduced in tamoxifen resistant breast cancer cells and re-expression inhibits cell proliferation and estrogen receptor alpha 66-regulated target gene transcription

Resistance to endocrine therapy is a major clinical problem in breast cancer. The role of ERalpha splice variants in endocrine resistance is largely unknown. We observed reduced protein expression of an N-terminally truncated ERalpha46 in endocrine-resistant LCC2, LCC9, and LY2 compared to MCF-7 breast cancer cells. Transfection of LCC9 and LY2 cells with hERalpha46 partially restored growth inhibition by TAM. Overexpression of hERalpha46 in MCF-7 cells reduced estradiol (E(2))-stimulated endogenous pS2, cyclin D1, nuclear respiratory factor-1 (NRF-1), and progesterone receptor transcription. Expression of oncomiR miR-21 was lower in TAM-resistant LCC9 and LY2 cells compared to MCF-7 cells. Transfection with ERalpha46 altered the pharmacology of E(2) regulation of miR-21 expression from inhibition to stimulation, consistent with the hypothesis that hERalpha46 inhibits ERalpha activity. Established miR-21 targets PTEN and PDCD4 were reduced in ERalpha46-transfected, E(2)-treated MCF-7 cells. In conclusion, ERalpha46 appears to enhance endocrine responses by inhibiting selected ERalpha66 responses.

Co-Inhibition of BCL-W and BCL2 Restores Antiestrogen Sensitivity through BECN1 and Promotes an Autophagy-Associated Necrosis

BCL2 family members affect cell fate decisions in breast cancer but the role of BCL-W (BCL2L2) is unknown. We now show the integrated roles of the antiapoptotic BCL-W and BCL2 in affecting responsiveness to the antiestrogen ICI 182,780 (ICI; Fulvestrant Faslodex), using both molecular (siRNA; shRNA) and pharmacologic (YC137) approaches in three breast cancer variants; MCF-7/LCC1 (ICI sensitive), MCF-7/LCC9 (ICI resistant), and LY2 (ICI resistant). YC137 inhibits BCL-W and BCL2 and restores ICI sensitivity in resistant cells. Co-inhibition of BCL-W and BCL2 is both necessary and sufficient to restore sensitivity to ICI, and explains mechanistically the action of YC137. These data implicate functional cooperation and/or redundancy in signaling between BCL-W and BCL2, and suggest that broad BCL2 family member inhibitors will have greater therapeutic value than targeting only individual proteins. Whereas ICI sensitive MCF-7/LCC1 cells undergo increased apoptosis in response to ICI following BCL-W±BCL2 co-inhibition, the consequent resensitization of resistant MCF-7/LCC9 and LY2 cells reflects increases in autophagy (LC3 cleavage; p62/SQSTM1 expression) and necrosis but not apoptosis or cell cycle arrest. Thus, de novo sensitive cells and resensitized resistant cells die through different mechanisms. Following BCL-W+BCL2 co-inhibition, suppression of functional autophagy by 3-methyladenine or BECN1 shRNA reduces ICI-induced necrosis but restores the ability of resistant cells to die through apoptosis. These data demonstrate the plasticity of cell fate mechanisms in breast cancer cells in the context of antiestrogen responsiveness. Restoration of ICI sensitivity in resistant cells appears to occur through an increase in autophagy-associated necrosis. BCL-W, BCL2, and BECN1 integrate important functions in determining antiestrogen responsiveness, and the presence of functional autophagy may influence the balance between apoptosis and necrosis.

The developmental protein HOXc11 as a mediator of endocrine resistance in breast cancer.

Abstract Abstract #3034 The nuclear receptor coactivator, SRC-1, has been implicated as an important mediator of resistance to endocrine therapy in breast cancer through work from our group and others. Using mass spectrometry we identified the homeobox protein HOXc11 as an interacting partner for the nuclear receptor coactivator SRC-1 in endocrine resistant breast cancer cells. Homeobox genes control cell differentiation. The processes of neoplasia and normal development are closely linked, therefore it is little surprise that increasing evidence implicates these genes in neoplasia development.
 We propose that the interaction between SRC-1 and HOXc11 plays an important role in the development of endocrine resistance. Our aim was to identify potential HOXc11/SRC-1 target genes and assess the clinical significance of these genes.
 Co-immunprecipitation assays confirmed the SRC-1 and HOXc11 interaction after treatment with tamoxifen for 2 hours in the endocrine resistant LY2 breast cancer cell line, an interaction that was not present in endocrine sensitive MCF-7 cells under the same treatment conditions. A target gene for HOXc11, S100β, has previously been identified in neuronal cells. Based on this we sought the presence of either protein at the promoter region for S100β. Chromatin immunoprecipitation (ChIP) assays in LY2 cells confirmed recruitment of both HOXc11 and SRC-1 to the promoter region of S100β after treatment with tamoxifen at both 45 minutes and 2 hours when compared to control.
 To assess the clinical significance of S100β, we used immunohistochemistry on a paraffin embedded tissue microarray to examine itÂs expression in a large cohort of breast cancer patients (n=560). S100β was a strong predictor of reduced disease free survival in patients receiving adjuvant endocrine treatment (Hazard ratio 5.82; p<0.0001). This also showed that expression of S100β was associated with expression of HOXc11 and SRC-1. Given that S100β is a secreted protein, we hypothesized that elevated levels could be detected in patients with breast cancer at time of diagnosis. Using an ELISA to measure S100β levels, we found elevated levels in one quarter of breast cancer patients, pre treatment, when compared to age matched controls.
 Investigation into non steroidal SRC-1 binding partners in endocrine resistant breast cancer identified the homeobox protein HOXc11. A target gene for the interaction between SRC-1 and HOXc11, S100β, is a secreted protein and raises the exciting possibility that it may offer potential as a biomarker for endocrine resistance in breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3034.

Regulation of COUP‐TFII expression in Tamoxifen‐ sensitive and resistant breast cancer cells.

Tamoxifen (TAM) is used for treatment and prevention of breast cancer. TAM competes with estradiol (E2) in binding estrogen receptors α and β (ERα and ER β) inhibiting proliferation. However, initially TAM-sensitive tumors become TAM-resistant leading to metastases. The mechanism of TAM resistance is unknown and biomarkers of TAM-response may be of use to monitor clinical response. One possible biomarker of TAM-response is the orphan nuclear receptor COUP-TFII which we found reduced in TAM-resistant breast cancer. We determined the effects of serum, phenol red, E2, 4-hydroxyTAM (4-OHT), ICI 182,780 (ICI), and a phytoestrogen resveratrol on cell proliferation and COUP-TFII expression in TAM- sensitive (MCF-7 and LCC1) and resistant (LCC9 and LY2) cell lines. Results showed MCF-7 and LCC1 cells are TAM-sensitive, LCC9 and LY2 cells are resistant to TAM and ICI. Resveratrol inhibited proliferation in all cells. Reduced serum inhibited proliferation of TAM-sensitive cells. Phenol red had estrogen agonist activity in a cell line-specific manner. siRNA reduction of ERα in MCF-7 cells did not affect COUP-TFII expression, but PD98059 inhibited E2-induced COUP-TFII expression. These results indicate COUP-TFII expression is not dependent on ERα in MCF-7 cells and suggest a possible role for ER β in regulating COUP-TFII expression. Supported by Susan G. Komen Breast Cancer Foundation grant BCTR0201438 to CMK.

Constitutive MEK/MAPK activation leads to p27(Kip1) deregulation and antiestrogen resistance in human breast cancer cells.

Antiestrogens, such as the drug tamoxifen, inhibit breast cancer growth by inducing cell cycle arrest. Antiestrogens require action of the cell cycle inhibitor p27(Kip1) to mediate G1 arrest in estrogen receptor-positive breast cancer cells. We report that constitutive activation of the mitogen-activated protein kinase (MAPK) pathway alters p27 phosphorylation, reduces p27 protein levels, reduces the cdk2 inhibitory activity of the remaining p27, and contributes to antiestrogen resistance. In two antiestrogen-resistant cell lines that showed increased MAPK activation, inhibition of the MAPK kinase (MEK) by addition of U0126 changed p27 phosphorylation and restored p27 inhibitory function and sensitivity to antiestrogens. Using antisense p27 oligonucleotides, we demonstrated that this restoration of antiestrogen-mediated cell cycle arrest required p27 function. These data suggest that oncogene-mediated MAPK activation, frequently observed in human breast cancers, contributes to antiestrogen resistance through p27 deregulation.

Expression of proangiogenic chemokine Gro 1 in low and high metastatic variants of Pam murine squamous cell carcinoma is differentially regulated by IL-1alpha, EGF and TGF-beta1 through NF-kappaB dependent and independent mechanisms.

We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in murine squamous cell carcinoma (SCC) with metastatic tumor progression. The enhanced expression of Gro-1 gene by SCC is regulated by activation of nuclear factor-kappaB (NF-kappaB), leading to accelerated tumor growth, angiogenesis and metastasis in vivo. In our study, we investigated the effect of the regulatory cytokines, IL-1alpha, EGF and TGF-beta1 on activation of NF-kappaB and Gro1 in primary and metastatic sublines of the murine SCC Pam 212. We found that Gro 1 expression could be induced by IL-1alpha or EGF in the low cytokine producing Pam 212 cells, but no significant induction was observed in high cytokine producing and metastatic LY-2 cells. Conditioned medium from LY-2 containing functional IL-1alpha induced Gro 1 expression in Pam 212 cells, which can be blocked by IL-1 receptor antagonist (IL-1RA). IL-1RA, however, had a minimal effect on constitutive Gro 1 production by LY-2 cells. TGF-beta1 suppressed constitutive as well as IL-1alpha and EGF-inducible Gro 1 production in both Pam 212 and LY-2 cells. IL-1alpha and EGF, but not TGF-beta1, were found to activate NF-kappaB in Pam 212, whereas none of the stimulants showed a significant effect on constitutive activation of NF-kappaB in LY-2 cells. Overexpression of a super repressor IkappaBalphaM in Pam 212 inhibited NF-kappaB binding activity, which led to impaired Gro 1 induction by IL-1alpha and EGF. These results demonstrate that IL-1alpha, EGF, and TGF-beta1 are important modulators of Gro 1 expression in SCC. Different responses to these modulators observed along with SCC metastatic progression may suggest a transition mechanism(s) for Gro 1 expression from host factor dependent to an independent stage involving NF-kappaB activation. Published 2001 Wiley-Liss, Inc.

Induction of human breast cancer cell apoptosis from G 2/M preceded by stimulation into the cell cycle by Z-1,1-dichloro-2,3-diphenylcyclopropane

We have shown previously that Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k.a. Analog II, A II) inhibits human breast cancer cell proliferation regardless of estrogen receptor status or estrogen sensitivity, and that its cellular targets include microtubules. In the present study, we investigated the apoptosis-inducing effects of A II. MCF-7, MCF-7/LY2, and MDA-MB-231 cells all showed nuclear fragmentation in response to 100 μM A II when stained with Hoechst 33342 and examined by fluorescence microscopy. Pulsed field gel electrophoretic analysis showed that each of the cell lines also developed specific high molecular weight DNA fragments: a low level of 1–2 Mb fragments appeared after 6 hr, while 30–50 kb fragments accumulated subsequently. At 24 hr of drug exposure, the majority of cells became nonadherent, and the 30–50 kb fragments were restricted to detached MCF-7 and MDA-MB-231 cells. Both adherent and detached MCF-7/LY2 cells exhibited these fragments. A previous study by single-color (propidium) flow cytometry demonstrated that A II blocks MDA-MB-231 cells in G 2/M of the cell cycle. More refined analyses in the present study showed this same result for MDA-MB-231 cells, but MCF-7 and MCF-7/LY2 cells did not reveal apparent drug-induced cell cycle block. A II demonstrated growth inhibitory, cell cycle-perturbing, and hypodiploidy-inducing activity against other human breast carcinoma lines, i.e. BT-20, CAMA-1, and SKBR-3, but no such actions in the non-tumorigenic, “normal” human breast epithelial line MCF-10A. Bromodeoxyuridine labeling and two-color flow cytometric analysis, however, suggested that A II caused stimulation into S phase, and that G 2/M was the phase of the cell cycle from which cells apoptosed. A II caused cell rounding, detachment from the growth matrix, and nuclear shrinkage and fragmentation in parallel with biochemical changes. Cycloheximide inhibited A II-induced cell death, indicating that its toxicity requires de novo protein synthesis.

Increased Sensitivity of the Antiestrogen-Resistant MCF-7/LY2 Human Breast Carcinoma Cell Line to Apoptosis Induced by the Novel Microtubule Stabilizing Agent (+)-Discodermolide

Abstract: (+)-Discodermolide is a sponge-derived natural product with the most potent microtubule stabilizing activity yet discovered. Its actions parallel that of the promising antibreast cancer agent paclitaxel despite the lack of any apparent similarities in the drugs' structures. To complement our previous studies on human breast cancer cells, we compared the effects of the two drugs against the estrogen receptor positive but tamoxifen-resistant MCF-7/LY2 line. Growth inhibition, cell, and nuclear morphological, electrophoretic, and flow cytometric analyses were performed. (+)-Discodermolide potently inhibited the growth of the cells (e.g., 48-hours IC50 of 1.5 nM) at concentrations similar to those observed with paclitaxel, and somewhat lower than the values observed previously with estrogen responsive MCF-7 cells and estrogen nonresponsive MDA-MB231 cells. (+)-Discodermolide-treated MCF-7/LY2 cells had condensed and highly fragmented nuclei, as well as micronuclei, suggesting mitotic block and the induction of apoptosis. Flow cytometric comparison of cells treated with either drug at 10 nM showed both caused accumulation into the G2/M portion of the cell cycle as well as induction of a pronounced hypodiploid cell population, with (+)-discodermolide yielding a greater effect. The timing and type of high molecular weight DNA fragmentation induced by the two agents was fully consistent with induction of apoptosis, again with (+)-discodermolide showing an advantage over paclitaxel in this regard. More extensive DNA fragmentation was noted in MCF-7/LY2 than has been observed in MCF-7 and MDA-MB231 cells. These in vitro results, coupled with those obtained previously, suggest that (+)-discodermolide might have promise as a new chemotherpeutic agent against breast cancers. In addition, its novel and synthetically approachable structure make (+)-discodermolide a promising lead compound for design and discovery of new microtubule stabilizing agents as alternatives to taxoids.

The anti-proliferative effect of suramin towards tamoxifen-sensitive and resistant human breast cancer cell lines in relation to expression of receptors for epidermal growth factor and insulin-like growth factor-I: Growth stimulation in the presence of tamoxifen

A significant proportion of breast cancer patients receiving tamoxifen therapy relapse during treatment following acquisition of tamoxifen-resistant or oestrogen-independent phenotypes. The mechanism behind this rapid progression to oestrogen autonomy is at present unclear and further treatment modalities are limited. Suramin represents a novel potential second line therapy. The mechanism of the antineoplastic activity of suramin is not completely understood, although the drug binds to many growth factors including epidermal growth factor and insulin-like growth factors and can also dissociate growth factors from their receptors. In this study we have related suramin sensitivity to the expression of receptors for epidermal growth factor and insulin-like growth factor-I in a number of breast cancer cell lines including lines resistant to tamoxifen. The anti-proliferative effects of suramin were investigated in two oestrogen dependent breast cancer lines (ZR-75-1 and MCF-7), oestrogen independent (ZR-PR-LT) and tamoxifen resistant (ZR-75-9a1) variants of ZR-75-1 and a tamoxifen resistant (LY2) variant of MCF-7. Full dose response curves were constructed and IC50 values determined for each cell line. Sensitivity to suramin was correlated with the level of expression of receptors for epidermal growth factor (EGFR) and insulin-like growth factor-I (IGFR). On observing stimulation of cell proliferation by suramin in the tamoxifen resistant cell lines in the presence of tamoxifen we also investigated the possible role of suramin sequestration of transforming growth factor-beta in mediating this effect. All cell lines exhibited a dose- and time-dependent response to suramin treatment. Tamoxifen resistant ZR-75-9a1 cells (day 6 IC50 85 micrograms ml-1) were more resistant to suramin than oestrogen independent ZR-PR-LT cells (day 6 IC50 45 micrograms ml-1), and the parent ZR-75-1 line (day 6 IC50 56 micrograms ml-1). Increased sensitivity to suramin was associated with increased expression of IGFR and decreased expression of EGFR. Tamoxifen resistant LY2 cells were significantly more sensitive to suramin (day 6 IC50 70 micrograms ml-1) than MCF-7 cells (day 6 IC50 350 micrograms ml-1). Both IGFR and EGFR expression by LY2 cells was lower than in the parent line. The antioestrogen-resistant ZR-75-9a1 and LY2 lines grown in the presence of 8 microM tamoxifen were growth stimulated by concentrations of the drug below 100 micrograms/ml. As growth stimulation observed in the presence of tamoxifen may have been due to suramin sequestration of tamoxifen induced TGF-beta 1 secretion we also investigated the response of the cells to this peptide in the presence and absence of suramin. All cell lines were growth inhibited by TGF-beta 1 except ZR-75-9a1 which was unresponsive. Responses to TGF-beta 1 were modified in the presence of 100 micrograms suramin ml-1 although TGF-beta 1 was unable to mimic the ability of tamoxifen to stimulate proliferation in the presence of suramin. These results suggest that for ZR-75-1 cells and variants, increased sensitivity to suramin is associated with an increase in expression of IGFR and a decrease in EGFR numbers. However, tamoxifen resistant LY2 cells, in which both IGFR and EGFR expression is reduced were considerably more sensitive than parental MCF-7 cells suggesting that there is no clear relationship between EGFR and IGFR expression and suramin sensitivity. The unexpected stimulation of cell proliferation of the tamoxifen resistant variants by suramin in the presence of tamoxifen could not be explained by suramin sequestration of transforming growth factor-beta and the mechanism of this interaction remains unclear.

Modulation of T-cell ontogeny by transplacental benzo(a)pyrene

Transplacental exposure to the carcinogen, benzo(a)pyrene BaP, leads to depressed immune function and increased tumor incidence in mice. This paper reports ontogenetic T-cell changes in BALB/c mice after exposure to BaP in utero. Monoclonal antibodies (MAbs) were produced to fetal liver T-cells (FLT) and newborn spleen (NBS) lymphocytes purified from offspring of pregnant BALB/c mice that were given one injection of BaP (150 mg/kg body weight) in mid-gestation (day 11–13). The MAbs reacted with two T-cell membrane antigens (FLT and NBS) found in fetal liver, neonatal and adult thymus and spleen. Lymphocytes of BaP-exposed 19-day fetuses showed decreased subpopulation frequencies ( P<0.05) in fetal liver total T-cells (from 56% to 16%), Lyl cells (from 33% to 9%), and Ly2 cells (from 56% to 1%) compared with untreated controls. In contrast, BaP increased the subpopulation frequencies ( P<0.05) in FLT cells in fetal liver (from 20% to 52%) and in newborn spleen (from 21% to 51%), and increased NBS cells in newborn spleen (from 24% to 59%). The increased frequency in FLT and NBS cells was due to their relative resistance to BaP toxicity and/or BaP-enhanced proliferation in the neonatal period. Compared with untreated controls, BaP treatment resulted in reduced numbers of T-cells in fetal liver and showed a selective toxicity for Ly1 cells (89% reduction) and Ly2 cells (99% reduction), whereas FLT cells were not reduced and NBS cells were reduced by 60%. Six-week-old juvenile mice exposed to BaP in utero showed recovery of total T-cells to control levels in spleen and thymus, but showed depletion ( P<0.01) in thymic FLT cells (from 81% to 12%) and in splenic NBS cells (from 55% to 16%). The monoclonal antibodies developed for this study recognize novel cellular changes in the murine immune system that are associated with transplacental BaP. The FLT and NBS antigens may be useful biomarkers for developmental immune dysfunctions in progeny exposed to BaP in utero.

Evaluation of estrogen receptor, antiestrogen binding sites and calmodulin for antiestrogen resistance of two clones derived from the MCF-7 breast cancer cell line

Estrogen receptor (ER), antiestrogen binding sites (AEBS) and calmodulin (CaM) are potential targets of antiestrogen (AE) action. To analyse further which of these targets are primarily involved in the antiproliferative activity of these drugs against human breast cancers, two cell clones, namely the RTx6 and LY-2 variants, selected from MCF-7 cells for their resistance to high doses of tamoxifen (TAM) and the Keoxifen (KEO) analog LY 117018, respectively, were studied for their sensitivity to hydroxytamoxifen (OH-TAM) and KEO as well as the strong calmodulin antagonist calmidazolium. The effects of these drugs on both cell growth and progesterone receptor (PgR) concentration were assessed. Binding properties for ER, AEBS and CaM of each compound were also measured. Our results confirmed that basal growth of RTx6 and LY-2 cells was more resistant to OH-TAM and KEO than parent MCF-7 cells, although both displayed a significant inhibition at the highest doses assessed. In regard to calmidazolium inhibition, each variant behaved as did the MCF-7 line indicating that a modification at the CaM level was not responsible for their lower sensitivity to AEs. Nor could the association of CaM to ER which did not differ among all cell lines. Resistance of these variants was not related to AEBS in view of the total lack of such sites in RTx6 cells. However, under estrogenic growth stimulation such sites may play some role, since LY-2 cells in the presence of estradiol displayed a real antiestrogen-resistant pattern while RTx6 cells were more sensitive than MCF-7 cells to OH-TAM. This property was not found in the antagonism against estradiol-induced PgR synthesis which was observed with each variant. Thus the PgR concentration of RTx6 cells was strongly down-regulated by OH-TAM and KEO and reduced in LY-2 cells to the same extent as in MCF-7 cells. All these observationsshow that AE resistance is not entirely related to ER mediated events and that alterations at the ER and CaM levels are unlikely to account for the lower AE sensitivity of the variants investigated.

Antiestrogen resistance in ER positive breast cancer cells.

Acquisition of the antiestrogen resistance by breast cancer cells in vivo may result from a variety of mechanisms. The main pathway appears to involve loss of estrogen receptor (ER) expression or selection for ER negative cells among heterogenous population of tumor cells. However, clinical data suggest that, in about 30% of the cases, antiestrogen resistance arises even in the presence of estrogen receptors. Postulated mechanisms leading to the latter phenotype include selection for variant receptor forms during treatment, development of novel metabolic pathways for the drug, loss of nuclear co-factors, or activation of signal transduction pathway that cross activate ER signals. We have used an in vitro experimental system utilizing LY-2 cell line, an ER positive and antiestrogen resistant MCF-7 cell variant, to study the mechanism of antiestrogen resistance in the presence of functional ER. Result from a complementation experiment suggests that LY-2 phenotype is a recessive trait. Cloning of the genetic defect in the LY-2 cells would provide further insight for the mechanism of antiestrogen resistance in ER positive breast cancer cells.

Contrasting effect of delta-9-tetrahydrocannabinol on IL-2 activity in spleen and lymph node cells of mice of different ages

The purpose of this study was to investigate the immunomodulatory effect of delta-9-tetrahydrocannabinol (THC) on spleen and lymph node cells of adult and young mice. When plant mitogen (Con A or PHA) stimulated cells were treated with varying doses of THC, the proliferation of spleen and lymph node cells of adult and young mice was suppressed. This suppression was found to be compatible with a decrease in number of Tac positive cells as well as a down-regulation of IL-2 activity. In contrast, when these cells were stimulated with anti-CD3 antibody in combination with THC, the pattern of the immunomodulation as well as the absolute effects of the drug differed with the age of the mice as well as the organ of origin of the cells. In this regard, anti-CD3 antibody stimulation in combination with low doses of THC stimulated proliferation of splenocytes obtained from adult mice. The stimulation was found to reflect an increase in the number of both suppressor/cytotoxic T cells (Ly2 cells) as well as Tac positive cells. IL-2 activity was found to be enhanced under these conditions. This upregulation of responsiveness was not seen in either lymph node cells of adult or young mice or in spleen cells of young mice. The THC modulating activity is directly related to its effect on both IL-2 activity and generation of Tac positive cells in spleen and lymph node cells of both adult and young mice. It appears, therefore, that not only age, but also lymphoid organ system differences are important factors in terms of considering the effects of drugs abuse.

Effects of interferon-gamma in T cell subsets of mice infected with Toxoplasma gondii

This study was performed to evaluate differences of T cell subsets according to the injection period of recombinant mouse interferon-gamma (IFN-gamma) in acute Toxoplasma gondii infection. Each mouse was infected intraperitoneally with 100 cysts of Beverley strain T. gondii, and injected with 5 x 10(4) units of IFN-gamma every other day two tmres. The percentage of Thy-1,2 cells and L3T4/Ly-2 cell ratio were significantly increased in the mice that received two doses of IFN-gamma on days 2 and 0 before infection, or days 0 and 2 after infection. The percentage of Ly-2 cells decreased in the IFN-gamma injected groups at the 3rd and 4th week after infection. The results suggest that administration of IFN-gamma to T. gondii-infected mice improves the changed population of T cell subsets to a normal state, especially when IFN-gamma was injected just after the infection.