RNA disruption is a widespread phenomenon associated with stress-induced cell death in tumour cells.

We have previously shown that neoadjuvant chemotherapy can induce the degradation of tumour ribosomal RNA (rRNA) in patients with advanced breast cancer, a phenomenon we termed "RNA disruption". Extensive tumour RNA disruption during chemotherapy was associated with a post-treatment pathological complete response and improved disease-free survival. The RNA disruption assay (RDA), which quantifies this phenomenon, is now being evaluated for its clinical utility in a large multinational clinical trial. However, it remains unclear if RNA disruption (i) is manifested across many tumour and non-tumour cell types, (ii) can occur in response to cell stress, and (iii) is associated with tumour cell death. In this study, we show that RNA disruption is induced by several mechanistically distinct chemotherapy agents and report that this phenomenon is observed in response to oxidative stress, endoplasmic reticulum (ER) stress, protein translation inhibition and nutrient/growth factor limitation. We further show that RNA disruption is dose- and time-dependent, and occurs in both tumourigenic and non-tumourigenic cell types. Northern blotting experiments suggest that the rRNA fragments generated during RNA disruption stem (at least in part) from the 28S rRNA. Moreover, we demonstrate that RNA disruption is reproducibly associated with three robust biomarkers of cell death: strongly reduced cell numbers, lost cell replicative capacity, and the generation of cells with a subG1 DNA content. Thus, our findings indicate that RNA disruption is a widespread phenomenon exhibited in mammalian cells under stress, and that high RNA disruption is associated with the onset of cell death.

Cystathionine gamma-lyase/H2 S signaling facilitates myogenesis under aging and injury condition.

Hydrogen sulfide (H2 S) can be endogenously produced and belongs to the class of signaling molecules known as gasotransmitters. Cystathionine gamma-lyase (CSE)-derived H2 S is implicated in the regulation of cell differentiation and the aging process, but the involvements of the CSE/H2 S system in myogenesis upon aging and injury have not been explored. In this study, we demonstrated that CSE acts as a major H2 S-generating enzyme in skeletal muscles and is significantly down-regulated in aged skeletal muscles in mice. CSE deficiency exacerbated the age-dependent sarcopenia and cardiotoxin-induced injury/regeneration in mouse skeletal muscle, possibly attributed to inefficient myogenesis. In contrast, supplement of NaHS (an H2 S donor) induced the expressions of myogenic genes and promoted muscle regeneration in mice. In vitro, incubation of myoblast cells (C2C12) with H2 S promoted myogenesis, as evidenced by the inhibition of cell cycle progression and migration, altered expressions of myogenic markers, elongation of myoblasts, and formation of multinucleated myotubes. Myogenesis was also found to upregulate CSE expression, while blockage of CSE/H2 S signaling resulted in a suppression of myogenesis. Mechanically, H2 S significantly induced the heterodimer formation between MEF2c and MRF4 and promoted the binding of MEF2c/MRF4 to myogenin promoter. MEF2c was S-sulfhydrated at both cysteine 361 and 420 in the C-terminal transactivation domain, and blockage of MEF2c S-sulfhydration abolished the stimulatory role of H2 S on MEF2c/MRF4 heterodimer formation. These findings support an essential role for H2 S in maintaining myogenesis, presenting it as a potential candidate for the prevention of age-related sarcopenia and treatment of muscle injury.

The RNA disruption assay is superior to conventional drug sensitivity assays in detecting cytotoxic drugs.

Conventional drug sensitivity assays used to screen prospective anti-cancer agents for cytotoxicity monitor biological processes associated with active growth and proliferation, used as proxies of cell viability. However, these assays are unable to distinguish between growth-arrested (but otherwise viable) cells and non-viable/dead cells. As a result, compounds selected based on the results of these assays may only be cytostatic, halting or slowing tumour progression temporarily, without tumour eradication. Because agents capable of killing tumour cells (cytotoxic drugs) are likely the most promising in the clinic, there is a need for drug sensitivity assays that reliably identify cytotoxic compounds that induce cell death. We recently developed a drug sensitivity assay, called the RNA disruption assay (RDA), which measures a phenomenon associated with tumour cell death. In this study, we sought to compare our assay's performance to that of current commonly used drug sensitivity assays (i.e, the clonogenic, the cell counting kit-8 and the Trypan blue exclusion assays). We found that RNA disruption occurred almost exclusively when total cell numbers decreased (cytotoxic concentrations), with little to no signal detected until cells had lost viability. In contrast, conventional assays detected a decrease in their respective drug sensitivity parameters despite cells retaining their viability, as assessed using a recovery assay. We also found that the RDA can differentiate between drug-sensitive and -resistant cells, and that it can identify agents capable of circumventing drug resistance. Taken together, our study suggests that the RDA is a superior drug discovery tool, providing a unique assessment of cell death.

RNA disruption indicates CHOP therapy efficacy in canine lymphoma.

BACKGROUND: Assessment of the efficacy of a multi-agent chemotherapy protocol in which cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) are administered in canine lymphoma is generally performed by physical measurement of lymph node diameter. However, no consistent correlation has been made with prognostic indicators and the length or absence of clinical remission based on lymph node size. RNA disruption measured mid-therapy has been correlated with increased disease-free survival in recent studies of human cancer and was assessed in this study of canine lymphoma patients. Fine needle aspirate samples were taken before treatment and at weeks 3, 6, and 11 of CHOP therapy. RNA was isolated from these samples and assessed using an Agilent Bioanalyzer. RNA disruption assay (RDA) analysis was performed on the data from the resulting electropherograms. RESULTS: An increased RNA disruption index (RDI) score was significantly associated with improved progression-free survival. CONCLUSIONS: Predicting the risk of early relapse during chemotherapy could benefit veterinary patients by reducing ineffective treatment and could allow veterinary oncologists to switch earlier to a more effective drug regimen.

Efflux inhibition by H2S confers sensitivity to doxorubicin-induced cell death in liver cancer cells.

AIMS: Hydrogen sulfide (H2S), an important gasotransmitter, is involved in a variety of cellular functions and pathophysiologic processes. Drug resistance due to alterations in drug trafficking and metabolism severely limits the effectiveness of cancer therapy. This study examined the role of H2S in drug resistance in liver cancer cells. MATERIALS AND METHODS: Human primary hepatocellular carcinoma cell line (HepG2) and doxorubicin (Dox)-resistant cells were used in this study. Cell survival was analyzed by MTT, Annexin V-FITC/propidium iodide staining and clonogenic assay. Western blotting was used for analysis of protein expression, and immunoprecipitation was used to determine interactions of LXR/RXR. KEY FINDINGS: The expression of H2S-generating enzyme cystathionine gamma-lyase (CSE) was inhibited by doxorubicin treatment in HepG2 cells, and H2S sensitized Dox-inhibited cell survival and colony formation. In addition, H2S promoted cellular retention of Dox by suppressing the expressions of ABCA1 and ABCG8. H2S significantly blocked Dox-induced heterodimer formation between LXRα and RXRß and attenuated the binding of LXRα/RXRß to the promoters of ABCA1 and ABCG8 genes. RXRß but not LXRα was S-sulfhydrated by H2S, and blockage of RXRß S-sulfhydration abolished the inhibitory role of H2S on LXRα/RXRß heterodimer formation. CSE expression was reduced in Dox-resistant cells in comparison with their parental cells, while H2S could reverse drug resistance in Dox-resistant cells. SIGNIFICANCE: Our study provides a novel solution for reversing drug resistance in cancer cells by targeting H2S signalling.

Alterations in estrogen signalling pathways upon acquisition of anthracycline resistance in breast tumor cells.

Intrinsic or acquired drug resistance is a major impediment to the successful treatment of women with breast cancer using chemotherapy. We have observed that MCF-7 breast tumor cells selected for resistance to doxorubicin or epirubicin (MCF-7DOX2 and MCF-7EPI cells, respectively) exhibited increased expression of several members of the aldo-keto reductase (AKR) gene family (in particular AKR1C3 and AKR1B10) relative to control MCF-7CC cells selected by propagation in the absence of drug. Normal cellular roles for the AKRs include the promotion of estrogen (E2) synthesis from estrone (E1) and the hydroxylation and detoxification of exogenous xenobiotics such as anthracycline chemotherapy drugs. While hydroxylation of anthracyclines strongly attenuates their cytotoxicity, it is unclear whether the enhanced AKR expression in the above anthracycline-resistant cells promotes E2 synthesis and/or alterations in E2 signalling pathways and whether such changes contribute to enhanced survival and anthracycline resistance. To determine the role of AKRs and E2 pathways in doxorubicin resistance, we examined changes in the expression of E2-related genes and proteins upon acquisition of doxorubicin resistance. We also assessed the effects of AKR overexpression or downregulation or the effects of activators or inhibitors of E2-dependent pathways on previously acquired resistance to doxorubicin. In this study we observed that the enhanced AKR expression upon acquisition of anthracycline resistance was, in fact, associated with enhanced E2 production. However, the expression of estrogen receptor α (ERα) was reduced by 2- to 5-fold at the gene transcript level and 2- to 20-fold at the protein level upon acquisition of anthracycline resistance. This was accompanied by an even stronger reduction in ERα phosphorylation and activity, including highly suppressed expression of two proteins under E2-dependent control (Bcl-2 and cyclin D1). The diminished Bcl-2 and cyclin D1 expression would be expected to reduce the growth rate of the cells, a hypothesis which was confirmed in subsequent cell proliferation experiments. AKR1C3 or AKR1B10 overexpression alone had no effect on doxorubicin sensitivity in MCF-7CC cells, while siRNA-mediated knockdown of AKR1C3 and/or AKR1B10 expression had no significant effect on sensitivity to doxorubicin in MCF-7DOX2 or MCF-7EPI cells. This suggested that enhanced or reduced AKR expression/activity is insufficient to confer anthracycline resistance or sensitivity to breast tumor cells, respectively. Rather, it would appear that AKR overexpression acts in concert with other proteins to confer anthracycline resistance, including reduced E2-dependent expression of both an important apoptosis inhibitor (Bcl-2) and a key protein associated with activation of cell cycle-dependent kinases (cyclin D1).

Role of autophagy and lysosomal drug sequestration in acquired resistance to doxorubicin in MCF-7 cells.

BACKGROUND: The roles and mechanisms involved in starvation-induced autophagy in mammalian cells have been extensively studied. However, less is known about the potential role for autophagy as a survival pathway in acquired drug resistance in cancer cells under nutrient-rich conditions. METHODS: We selected MCF-7 breast tumor cells for survival in increasing concentrations of doxorubicin and assessed whether the acquisition of doxorubicin resistance was accompanied by changes in doxorubicin and lysosome localization and the activation of autophagy, as assessed by laser scanning confocal microscopy with or without immunohistochemical approaches. The ultrastructure of cells was also viewed using transmission electron microscopy. Cellular levels of autophagy and apoptosis-related proteins were assessed by immunoblotting techniques, while protein turnover was quantified using a flux assay. RESULTS: As cells acquired resistance to doxorubicin, the subcellular location of the drug moved from the nucleus to the perinuclear region. The location of lysosomes and autophagosomes also changed from being equally distributed throughout the cytoplasm to co-localizing with doxorubicin in the perinuclear region. There was an apparent temporal correlation between the acquisition of doxorubicin resistance and autophagy induction, as measured by increases in monodansylcadaverine staining, LC3-II production, and co-localization of LAMP1 and LC3-II immunofluorescence. Electron microscopy revealed an increase in cytoplasmic vacuoles containing mitochondria and other cellular organelles, also suggestive of autophagy. Consistent with this view, a known autophagy inhibitor (chloroquine) was highly effective in restoring doxorubicin sensitivity in doxorubicin-resistant cells. Moreover, this induction of autophagy correlated temporally with increased expression of the selective cargo receptor p62, which facilitates the delivery of doxorubicin-damaged mitochondria and other organelles to autophagosomes. Finally, we suggest that autophagy associated with doxorubicin resistance may be distinct from classical starvation-induced autophagy, since Beclin 1 and Atg7 expression did not change upon acquisition of doxorubicin resistance, nor did recombinant Bcl2 overexpression or an Atg7 knockdown alter doxorubicin cytotoxicity. CONCLUSION: Taken together, our findings suggest that doxorubicin resistance in MCF-7 breast cancer cells is mediated, at least in part, by the activation of autophagy, which may be distinct from starvation-induced autophagy.

RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines.

BACKGROUND: Cellular stressors and apoptosis-inducing agents have been shown to induce ribosomal RNA (rRNA) degradation in eukaryotic cells. Recently, RNA degradation in vivo was observed in patients with locally advanced breast cancer, where mid-treatment tumor RNA degradation was associated with complete tumor destruction and enhanced patient survival. However, it is not clear how widespread chemotherapy induced "RNA disruption" is, the extent to which it is associated with drug response or what the underlying mechanisms are. METHODS: Ovarian (A2780, CaOV3) and breast (MDA-MB-231, MCF-7, BT474, SKBR3) cancer cell lines were treated with several cytotoxic chemotherapy drugs and total RNA was isolated. RNA was also prepared from docetaxel resistant A2780DXL and carboplatin resistant A2780CBN cells following drug exposure. Disruption of RNA was analyzed by capillary electrophoresis. Northern blotting was performed using probes complementary to the 28S and 18S rRNA to determine the origins of degradation bands. Apoptosis activation was assessed by flow cytometric monitoring of annexin-V and propidium iodide (PI) binding to cells and by measuring caspase-3 activation. The link between apoptosis and RNA degradation (disruption) was investigated using a caspase-3 inhibitor. RESULTS: All chemotherapy drugs tested were capable of inducing similar RNA disruption patterns. Docetaxel treatment of the resistant A2780DXL cells and carboplatin treatment of the A2780CBN cells did not result in RNA disruption. Northern blotting indicated that two RNA disruption bands were derived from the 3'-end of the 28S rRNA. Annexin-V and PI staining of docetaxel treated cells, along with assessment of caspase-3 activation, showed concurrent initiation of apoptosis and RNA disruption, while inhibition of caspase-3 activity significantly reduced RNA disruption. CONCLUSIONS: Supporting the in vivo evidence, our results demonstrate that RNA disruption is induced by multiple chemotherapy agents in cell lines from different tissues and is associated with drug response. Although present, the link between apoptosis and RNA disruption is not completely understood. Evaluation of RNA disruption is thus proposed as a novel and effective biomarker to assess response to chemotherapy drugs in vitro and in vivo.

A phase I/II trial of epirubicin and docetaxel in locally advanced breast cancer (LABC) on 2-weekly or 3-weekly schedules: NCIC CTG MA.22.

This phase I/II neoadjuvant trial (ClinicalTrials.gov identifier NCT00066443) determined maximally-tolerated doses (MTD), dose-limiting toxicities, response-to-therapy, and explored the role of novel response biomarkers. MA.22 accrued T3N0, any N2 or N3, and T4 breast cancer patients. Treatment was 6 cycles of 3-weekly (Schedule A; N = 47) or 8 cycles of 2-weekly (Schedule B; N = 46) epirubicin/docetaxel chemotherapy in sequential phase I/II studies, with growth factor support. In phase I of each schedule, MTDs were based on DLT. In phase II, clinical responses (CR/PR) and pathologic complete responses (pCR) were assessed. Tumor biopsy cores were obtained pre-, mid-, and post-treatment: 3 for pathologic assessment; 3 for microarray studies. DLT for Schedule A was febrile neutropenia at 105 mg/m(2) epirubicin and 75 mg/m(2) docetaxel; for schedule B, it was fatigue at 75 mg/m(2) for both agents. Phase II doses were 90 mg/m(2) epirubicin/75 mg/m(2) docetaxel for Schedule A and 60 mg/m(2) (both agents) for Schedule B. Schedule A CR/PR and pCR rates were 90 and 10 %, with large reductions in tumor RNA content and integrity following treatment; Schedule B results were 93 and 0 %, with smaller reductions in RNA quality. Pre-treatment expression of several genes was associated with clinical response, including those within a likely amplicon at 17q12 (ERBB2, TCAP, GSDMB, and PNMT). The combination regimens had acceptable toxicity, good clinical response, induction of changes in tumor RNA content and integrity. Pre-treatment expression of particular genes was associated with clinical responses, including several near 17q12, which with ERBB2, may better identify chemoresponsiveness.

Tumor RNA disruption predicts survival benefit from breast cancer chemotherapy.

In a prior substudy of the CAN-NCIC-MA.22 clinical trial (ClinicalTrials.gov identifier NCT00066443), we observed that neoadjuvant chemotherapy reduced tumor RNA integrity in breast cancer patients, a phenomenon we term "RNA disruption." The purpose of the current study was to assess in the full patient cohort the relationship between mid-treatment tumor RNA disruption and both pCR post-treatment and, subsequently, disease-free survival (DFS) up to 108 months post-treatment. To meet these objectives, we developed the RNA disruption assay (RDA) to quantify RNA disruption and stratify it into 3 response zones of clinical importance. Zone 1 is a level of RNA disruption inadequate for pathologic complete response (pCR); Zone 2 is an intermediate level, while Zone 3 has high RNA disruption. The same RNA disruption cut points developed for pCR response were then utilized for DFS. Tumor RDA identified >fourfold more chemotherapy non-responders than did clinical response by calipers. pCR responders were clustered in RDA Zone 3, irrespective of tumor subtype. DFS was about 2-fold greater for patients with tumors in Zone 3 compared to Zone 1 patients. Kaplan-Meier survival curves corroborated these findings that high tumor RNA disruption was associated with increased DFS. DFS values for patients in zone 3 that did not achieve a pCR were similar to that of pCR recipients across tumor subtypes, including patients with hormone receptor positive tumors that seldom achieve a pCR. RDA appears superior to pCR as a chemotherapy response biomarker, supporting the prospect of its use in response-guided chemotherapy.

RNA Disruption and Drug Response in Breast Cancer Primary Systemic Therapy.

BACKGROUND: As there is now evidence that switching clinical nonresponders early in primary systemic therapy to alternate treatment regimens can enhance survival in some breast cancer patients, the need for a robust intermediate endpoint that can guide treatment response across all tumor subtypes is urgent. Recently, chemotherapy drugs have been shown to induce a decrease in RNA quality in tumor cells from breast cancer biopsies in some patients at midtherapy, and that this has been associated with subsequent achievement of pathological complete response (pCR). The decrease in RNA quality has been shown to be associated with RNA disruption; aberrant RNA bands visualized by RNA electrophoresis have been associated with subsequent tumor cell death. The objectives of these studies are to show that a new assay based on induction of RNA disruption in tumor cells by chemotherapy can stratify at midtherapy, pCR responders from non-pCR responders irrespective of clinical response and to present early evidence that clinically useful RNA disruption can be detected as early as 14 days after initiation of treatment. METHODS: RNA disruption in tumor cells was quantified by analysis of the RNA electrophoresis banding pattern and expressed as an RNA disruption index (RDI). To develop the RNA disruption assay (RDA), RDI was correlated with clinical outcome (pCR) from the NCIC-CTG MA.22 breast cancer clinical trial (ClinicalTrials.gov NCT00066443). RDA Zones were established by stratifying patients using RDI values into Zone 1, Zone 2, and Zone 3. Zone 3 included seven out of eight pCR responders, whereas Zone 1 contained no pCR responders. An intermediate zone (Zone 2) was established which contained one pCR. Subsequently, to determine early drug response, RNA disruption was examined by RDI after 14 days exposure to trastuzumab, zoledronic acid, or letrozole + cyclophosphamide ± sorafenib therapy. RESULTS: In MA.22, RDA stratified 23 of 85 patients in Zone 1 as pCR nonresponders, 24 patients in Zone 2, an intermediate zone, and 38 patients in Zone 3, pCR responders and non-pCR patients who share RDI comparable to those achieving pCR. In the early response studies, after 14 days exposure to chemotherapy, some RNA disruption as measured by RDI elevation could be detected in 3/12 trastuzumab, 7/15 zoledronic acid, 5/29 letrozole + cyclophosphamide, and 5/23 letrozole + cyclophosphamide + sorafenib patients. CONCLUSIONS: RDA is a novel intermediate endpoint that has promise for clinical utility for breast cancers early in response-guided primary systemic therapy.

Distinct genetic alterations occur in ovarian tumor cells selected for combined resistance to carboplatin and docetaxel.

BACKGROUND: Current protocols for the treatment of ovarian cancer include combination chemotherapy with a platinating agent and a taxane. However, many patients experience relapse of their cancer and the development of drug resistance is not uncommon, making successful second line therapy difficult to achieve. The objective of this study was to develop and characterize a cell line resistant to both carboplatin and docetaxel (dual drug resistant ovarian cell line) and to compare this cell line to cells resistant to either carboplatin or docetaxel. METHODS: The A2780 epithelial endometrioid ovarian cancer cell line was used to select for isogenic carboplatin, docetaxel and dual drug resistant cell lines. A selection method of gradually increasing drug doses was implemented to avoid clonal selection. Resistance was confirmed using a clonogenic assay. Changes in gene expression associated with the development of drug resistance were determined by microarray analysis. Changes in the expression of selected genes were validated by Quantitative Real-Time Polymerase Chain Reaction (QPCR) and immunoblotting. RESULTS: Three isogenic cell lines were developed and resistance to each drug or the combination of drugs was confirmed. Development of resistance was accompanied by a reduced growth rate. The microarray and QPCR analyses showed that unique changes in gene expression occurred in the dual drug resistant cell line and that genes known to be involved in resistance could be identified in all cell lines. CONCLUSIONS: Ovarian tumor cells can acquire resistance to both carboplatin and docetaxel when selected in the presence of both agents. Distinct changes in gene expression occur in the dual resistant cell line indicating that dual resistance is not a simple combination of the changes observed in cell lines exhibiting single agent resistance.

Role of aldo-keto reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization.

BACKGROUND: Since proteins involved in chemotherapy drug pharmacokinetics and pharmacodynamics have a strong impact on the uptake, metabolism, and efflux of such drugs, they likely play critical roles in resistance to chemotherapy drugs in cancer patients. METHODS: To investigate this hypothesis, we conducted a whole genome microarray study to identify difference in the expression of genes between isogenic doxorubicin-sensitive and doxorubicin-resistant MCF-7 breast tumour cells. We then assessed the degree of over-representation of doxorubicin pharmacokinetic and pharmacodynamic genes in the dataset of doxorubicin resistance genes. RESULTS: Of 27,958 Entrez genes on the array, 7.4 per cent or 2,063 genes were differentially expressed by ≥ 2-fold between wildtype and doxorubicin-resistant cells. The false discovery rate was set at 0.01 and the minimum p value for significance for any gene within the "hit list" was 0.01. Seventeen and 43 per cent of doxorubicin pharmacokinetic genes were over-represented in the hit list, depending upon whether the gene name was identical or within the same gene family, respectively. The most over-represented genes were within the 1C and 1B families of aldo-keto reductases (AKRs), which convert doxorubicin to doxorubicinol. Other genes convert doxorubicin to other metabolites or affect the influx, efflux, or cytotoxicity of the drug. In further support of the role of AKRs in doxorubicin resistance, we observed that, in comparison to doxorubicin, doxorubincol exhibited dramatically reduced cytotoxicity, reduced DNA-binding activity, and strong localization to extra nuclear lysosomes. Pharmacologic inhibition of the above AKRs in doxorubicin-resistant cells increased cellular doxorubicin levels, restored doxorubicin cytotoxicity and re-established doxorubicin localization to the nucleus. The properties of doxorubicinol were unaffected. CONCLUSIONS: These findings demonstrate the utility of using curated pharmacokinetic and pharmacodynamic knowledge bases to identify highly relevant genes associated with doxorubicin resistance. The induction of one or more of these genes was found to be correlated with changes in the drug's properties, while inhibiting one specific class of these genes (the AKRs) increased cellular doxorubicin content and restored drug DNA binding, cytotoxicity, and subcellular localization.

Antibody responses following vaccination versus infection in a porcine circovirus-type 2 (PCV2) disease model show distinct differences in virus neutralization and epitope recognition.

Porcine circovirus associated disease (PCVAD) encompasses a group of syndromes linked to infection with porcine circovirus type 2 (PCV2). Based on the hypothesis that the immune responses to vaccination versus infection are quantitatively and qualitatively different, the objective of this study was to evaluate immunity, virus replication and disease protection in pigs vaccinated with PCV2 capsid protein (CP) and during infection. The disease model included dual infection with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV), a virus known to enhance disease progression and severity. The principal effect of PRRSV infection was to increase peak PCV2 viremia by almost 40-fold; however, PCV2 failed to show a reciprocal effect on PRRSV. In vaccinated pigs, there was no evidence of disease or PCV2 replication following dual virus challenge. Immunity following vaccination favored PCV2 neutralizing activity; whereas, PCV2 infection and disease produced high levels of non-neutralizing antibody, primarily directed against a polypeptide in the C-terminal region of CP. These results support the notion that the magnitude of the total antibody response cannot be used as a measure of protective immunity. Furthermore, protection versus disease lies in the immunodominance of specific epitopes. Epitope specificity should be taken into consideration when designing PCV2 vaccines.

Alterations in tumor necrosis factor signaling pathways are associated with cytotoxicity and resistance to taxanes: a study in isogenic resistant tumor cells.

INTRODUCTION: The taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance. METHODS: MCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR). RESULTS: MCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways. CONCLUSIONS: We report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance.

Association of low tumor RNA integrity with response to chemotherapy in breast cancer patients.

The CAN-NCIC-MA22 phase I/II clinical trial evaluated women with locally advanced or inflammatory breast cancer treated with epirubicin and docetaxel at 2 or 3 weekly intervals in sequential cohorts. The relationship between various biomarkers and treatment response was assessed. Breast biopsy cores were obtained from 50 patients pre-, mid-, and post-treatment. Immunohistochemical staining was performed to determine baseline levels of estrogen receptor (ER), progesterone receptor (PR), Her2/Neu protein (HER2), and topoisomerase II (Topo 2),expressed as percent positive stain. Tumor RNA integrity(RIN) and tumor cellularity were measured pre-, mid- and post-treatment by capillary electrophoresis and light microscopy after hematoxylin/eosin staining, respectively.Associations between 1) maximum RIN and 2) tumor cellularity at the three time points with baseline levels of ER,PR, Her2, and topo II were assessed using Spearman and Pearson correlation coefficients. Associations between RIN and tumor cellularity with chemotherapy dose level orpathologic response were assessed using one-way ANOVA.In this study, we observed that low mid-treatment maximum RIN (but not tumor cellularity) was associated with high chemotherapy drug dose level (P = 0.05) and eventual pathologic complete response (pCR) (P = 0.01). Posttreatment,low maximum RIN was found to be associated with low tumor cellularity (P = 0.004), and low tumor cellularity with pCR (P = 0.01). Post-treatment tumor cellularity was lowest in patients with tumors having high baseline PR levels (P = 0.05). The association of midtreatment RIN with drug dose level and with pCR suggests that tumor RIN may represent an important new biomarker for measuring response to chemotherapy in breast cancer patients.

Induction of 1C aldoketoreductases and other drug dose-dependent genes upon acquisition of anthracycline resistance.

OBJECTIVES: Recent studies suggest that tumor cells overexpressing aldoketoreductases (AKRs) exhibit increased resistance to DNA damaging agents such as anthracyclines. AKRs may induce resistance to the anthracycline doxorubicin by catalyzing its conversion to the less toxic 13-hydroxy metabolite doxorubicinol. However, it has not been established whether during selection for anthracycline resistance, AKR overexpression in tumor cells can be correlated with the onset or magnitude of drug resistance and with appreciable conversion of anthracyclines to 13-hydroxy metabolites. METHODS AND FINDINGS: Through microarray and quantitative polymerase chain reaction studies involving rigid selection criteria and both correlative discriminate statistics and time-course models, we have identified several genes whose expression can be correlated with the onset and/or magnitude of anthracycline resistance, including AKR1C2 and AKR1C3. Also associated with the onset or magnitude of anthracycline resistance were genes involved in drug transport (ABCB1, ABCC1), cell signaling and transcription (RDC1, CXCR4), cell proliferation or apoptosis (BMP7, CAV1), protection from reactive oxygen species (AKR1C2, AKR1C3, FTL, FTH, TXNRD1, MT2A), and structural or immune system proteins (IFI30, STMN1). As expected, doxorubicin-resistant and epirubicin-resistant cells exhibited higher levels of doxorubicinol than wild-type cells, although at insufficient levels to account for significant drug resistance. Nevertheless, an inhibitor of Akr1c2 (5beta-cholanic acid) almost completely restored sensitivity to doxorubicin in ABCB1-deficient doxorubicin-resistant cells, while having no effect on ABCB1-expressing epirubicin-resistant cells. CONCLUSION: Taken together, we show for the first time that a variety of genes (particularly redox genes such as AKR1C2 and AKR1C3) can be temporally and causally correlated with the acquisition of anthracycline resistance in breast tumor cells.

Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance.

BACKGROUND: Anthracyclines and taxanes are commonly used in the treatment of breast cancer. However, tumor resistance to these drugs often develops, possibly due to overexpression of drug transporters. It remains unclear whether drug resistance in vitro occurs at clinically relevant doses of chemotherapy drugs and whether both the onset and magnitude of drug resistance can be temporally and causally correlated with the enhanced expression and activity of specific drug transporters. To address these issues, MCF-7 cells were selected for survival in increasing concentrations of doxorubicin (MCF-7DOX-2), epirubicin (MCF-7EPI), paclitaxel (MCF-7TAX-2), or docetaxel (MCF-7TXT). During selection cells were assessed for drug sensitivity, drug uptake, and the expression of various drug transporters. RESULTS: In all cases, resistance was only achieved when selection reached a specific threshold dose, which was well within the clinical range. A reduction in drug uptake was temporally correlated with the acquisition of drug resistance for all cell lines, but further increases in drug resistance at doses above threshold were unrelated to changes in cellular drug uptake. Elevated expression of one or more drug transporters was seen at or above the threshold dose, but the identity, number, and temporal pattern of drug transporter induction varied with the drug used as selection agent. The pan drug transporter inhibitor cyclosporin A was able to partially or completely restore drug accumulation in the drug-resistant cell lines, but had only partial to no effect on drug sensitivity. The inability of cyclosporin A to restore drug sensitivity suggests the presence of additional mechanisms of drug resistance. CONCLUSION: This study indicates that drug resistance is achieved in breast tumour cells only upon exposure to concentrations of drug at or above a specific selection dose. While changes in drug accumulation and the expression of drug transporters does occur at the threshold dose, the magnitude of resistance cannot be attributed solely to changes in drug accumulation or the activity of drug transporters. The identities of these additional drug-transporter-independent mechanisms are discussed, including their likely clinical relevance.

Gene expression profiles as biomarkers for the prediction of chemotherapy drug response in human tumour cells.

Genome profiling approaches such as cDNA microarray analysis and quantitative reverse transcription polymerase chain reaction are playing ever-increasing roles in the classification of human cancers and in the discovery of biomarkers for the prediction of prognosis in cancer patients. Increasing research efforts are also being directed at identifying set of genes whose expression can be correlated with response to specific drugs or drug combinations. Such genes hold the prospect of tailoring chemotherapy regimens to the individual patient, based on tumour or host gene expression profiles. This review outlines recent advances and challenges in using genome profiling for the identification of tumour or host genes whose expression correlates with response to chemotherapy drugs both in vitro and in clinical studies. Genetic predictors of response to a variety of anticancer agents are discussed, including the anthracyclines, taxanes, topoisomerase I and II inhibitors, nucleoside analogs, alkylating agents, and vinca alkaloids.

Cross-resistance studies of isogenic drug-resistant breast tumor cell lines support recent clinical evidence suggesting that sensitivity to paclitaxel may be strongly compromised by prior doxorubicin exposure.

Less than half of breast cancer patients respond to second-line chemotherapy with paclitaxel after failing treatment with anthracyclines such as doxorubicin. A recent clinical trial by Paridaens et al. [J. Clin. Oncol. 18 : 724-733, 2000] examined whether patients may derive a better clinical benefit if paclitaxel was administered before doxorubicin. While overall survival was similar regardless of the order of drug administration, a >4-fold reduction in the response rate to paclitaxel was observed after late crossover from doxorubicin, compared to the response rate to doxorubicin after late crossover from paclitaxel. This may be related to differences in the ability of the drugs to induce cross-resistance to each other. To test this hypothesis, we examined whether isogenic breast tumor cells selected for resistance to doxorubicin exhibit greater cross-resistance to paclitaxel and other drugs than identical cells selected for resistance to paclitaxel. We found that cells selected for resistance to paclitaxel showed strong resistance (>/=40-fold) to paclitaxel and docetaxel, with little cross-resistance (4-fold) to doxorubicin. In contrast, cells selected for resistance to doxorubicin exhibited 50-fold resistance to doxorubicin and a dramatic 4700-fold and 14,600-fold cross-resistance to paclitaxel and docetaxel, respectively. Doxorubicin-resistant cells exhibited higher P-glycoprotein and breast cancer resistance protein (BCRP) levels than paclitaxel-resistant cells. In addition, procaspase-9 was strongly downregulated in doxorubicin-resistant cells but not in paclitaxel-resistant cells. These differences may account for the contrasting cross-resistance profiles observed for the two cell lines and may help to explain why treatment of breast cancer patients with paclitaxel appears to be compromized by prior doxorubicin exposure.