Simona Mozzetti

Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample

We describe a semiquantitative RT-PCR protocol optimized in our laboratory to extract RNA from as little as 10,000 cells and to measure the expression levels of several target mRNAs from each sample. This procedure was optimized on the human erythroleukemia cell line TF-1 but was successfully used on primary cells and on different cell lines. We describe the detailed procedure for the analysis of Bcl-2 levels. Aldolase A was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. As for all quantitative techniques, great care must be taken in all optimization steps: the necessary controls to ensure a rough quantitative (semi-quantitative) analysis are described here, together with an example from a study on the effects of TGF-β1 in TF-1 cells.

Class III β-Tubulin Overexpression Is a Marker of Poor Clinical Outcome in Advanced Ovarian Cancer Patients

Purpose: Overexpression of β III tubulin has been involved in paclitaxel resistance in several experimental models. We investigated the role of β III tubulin as predictor of clinical outcome in ovarian cancer patients given platinum/paclitaxel treatment. We also investigated whether β III tubulin expression could be modified after the selective pressure represented by chemotherapy in vivo. Experimental Design: The study was designed to include a series of consecutive ovarian cancer patients with unresectable disease at time of first surgery, who underwent interval debulking surgery with pathologic assessment of response to treatment with platinum/paclitaxel chemotherapy. Immunostaining was done on formalin-fixed, paraffin-embedded tissue sections from pretreatment and posttreatment tissue biopsies by using the polyclonal rabbit anti–class III β-tubulin antibody. Results: β III Tubulin immunoreaction was observed in 51 of 62 (82.2%) cases. β III Tubulin positivity was neither associated with clinicopathologic variables nor with pathologic response to chemotherapy. Significantly lower percentages of β III tubulin positivity were observed in posttreatment (range, 5-80%; median, 20%) versus pretreatment (range 10-100%; median, 40%) tissue biopsies (P = 0.0011). Cases with high β III tubulin expression showed a worse overall survival with respect to cases with low β III tubulin expression (median overall survival, 25 versus 46 months; P = 0.002). Multivariate analysis showed that high content of β III tubulin remains independently associated with a worse prognosis. Conclusions: Assessment of β III tubulin could be useful to identify poor prognosis ovarian cancer patients candidates to more aggressive and/or targeted therapy.

Paclitaxel Directly Binds to Bcl-2 and Functionally Mimics Activity of Nur77

We reported previously that Bcl-2 is paradoxically down-regulated in paclitaxel-resistant cancer cells. We reveal here that paclitaxel directly targets Bcl-2 in the loop domain, thereby facilitating the initiation of apoptosis. Molecular modeling revealed an extraordinary similarity between the paclitaxel binding sites in Bcl-2 and beta-tubulin, leading us to speculate that paclitaxel could be mimetic of an endogenous peptide ligand, which binds both proteins. We tested the hypothesis that paclitaxel mimics Nur77, which, like paclitaxel, changes the function of Bcl-2. This premise was confirmed by Nur77 interacting with both paclitaxel targets (Bcl-2 and beta-tubulin) and a peptide sequence mimicking the Nur77 structural region, thus reproducing the paclitaxel-like effects of tubulin polymerization and opening the permeability transition pore channel in mitochondria. This discovery could help in the development of novel anticancer agents with nontaxane skeleton as well as in identifying the clinical subsets responsive to paclitaxel-based therapy.

The seco-taxane IDN5390 is able to target class III beta-tubulin and to overcome paclitaxel resistance.

A prominent mechanism of drug resistance to taxanes is the overexpression of class III β-tubulin. The seco-taxane IDN5390 was chosen for its selective activity in paclitaxel-resistant cells with an overexpression of class III β-tubulin. Moreover, the combined treatment paclitaxel/IDN5390 yielded a strong synergism, which was also evident in cell-free tubulin polymerization assays. In the presence of an anti-class III β-tubulin as a blocking antibody, tubulin polymerization induced by paclitaxel and IDN5390 was enhanced and not affected, respectively, whereas synergism was abolished, thereby indicating that IDN5390 activity is not modulated by class III β-tubulin levels. Such properties can be explained by taking into consideration the composition of class III β-tubulin paclitaxel binding site; in fact, Ser277 interacting with paclitaxel C group in class I is replaced by an Arginine in class III. IDN5390 that has an open and flexible C ring and an acidic α-unsaturated enol-keton moiety better fits with class III β-tubulin than paclitaxel at the binding site. Taking altogether, these findings indicate that the concomitant treatment IDN5390/paclitaxel is able to successfully target class I and III β-tubulin and the combined use of two taxanes with diverse spectrum activity against tubulin isotypes could represent a novel approach to overcome paclitaxel resistance.

CD34+/CD105+ cells are enriched in primitive circulating progenitors residing in the G0 phase of the cell cycle and contain all bone marrow and cord blood CD34+/CD38low/− precursors

A subset of circulating CD34+ cells was found to express CD105 antigen. Sorting experiments showed that most granulocyte–macrophage colony‐forming units (GM‐CFU) and burst‐forming units — erythroid (BFU‐E) were retained in the CD34+/CD105− fraction, whereas rare GM‐CFU/BFU‐E were generated from CD34+/CD105+ cells. Megakaryocytic aggregates were entirely retained in the CD34+/CD105+ fraction. Neutralizing doses of an anti‐TGF‐β1 antibody demonstrated CD34+/CD105+ cells capable of colony‐forming activity without any significant effect on CD34+/CD105− cells. Cloning of secondary colonies revealed that CD34+/CD105+ cells had a significantly higher secondary cloning efficiency than CD34+/CD105− cells. CD34+/CD105+ cells had a significantly higher long‐term culture‐initiating cell (LTC‐IC) frequency than CD34+/CD105− cells. Kinetic analysis showed that 75% of CD34+/CD105+ cells consisted of DNA 2n G0Ki‐67− cells whereas 82% of CD34+/CD105− were DNA 2n G1Ki‐67+ cells, and this latter subset showed a RNA content consistently higher than CD34+/CD105+ cells. CD34+/CD105+ progenitors were CD25+, whereas CD34+/CD105− contained a small CD25+ subset. Three‐colour analysis of bone marrow and cord blood CD34+ cells demonstrated that all the CD34+/CD38low/− primitive precursors were contained in CD34+/CD105+ cells. Extensive characterization of these CD105+ precursors indicated that they have biological properties associated with primitive haematopoietic precursors.

Molecular Mechanisms of Patupilone Resistance

Patupilone is an epothilone in advanced clinical development that has shown promising efficacy in heavily pretreated patients. This study aimed at characterizing the mechanisms of patupilone activity in resistant patients. To this end, we generated patupilone-resistant cells using two cellular models, the first characterized by high chemosensitivity and low class III beta-tubulin (TUBB3) expression (A2780), and the second by low chemosensitivity and high TUBB3 expression (OVCAR-3). The obtained cell lines were named EPO3 and OVCAR-EPO, respectively. The same selection procedure was done in A2780 cells to generate a paclitaxel-resistant cell line (TAX50). Factors of resistance are expected to increase in the drug-resistant cell lines, whereas factors of drug sensitivity will be down-regulated. Using this approach, we found up-regulation of TUBB3 in TAX50, but not EPO3, cells, showing that TUBB3 mediates the resistance to paclitaxel but not to patupilone. Moreover, TUBB3 was a factor of patupilone sensitivity because OVCAR-EPO cells exhibited a dramatic reduction of TUBB3 and a concomitant sensitization to hypoxia and cisplatin-based chemotherapy. To identify the mechanisms underlying patupilone resistance, tubulin genes were sequenced, thereby revealing that a prominent mechanism of drug resistance is represented by point mutations in class I beta-tubulin. Overall, these results suggest that paclitaxel and patupilone have nonoverlapping mechanisms of resistance, thus allowing the use of patupilone for those patients relapsing after paclitaxel-based chemotherapy. Furthermore, patupilone represents a promising first-line option for the treatment of high-risk ovarian cancer patients, who exhibit high TUBB3 levels and poor response to standard paclitaxel-platin chemotherapy.

Trichostatin A up-regulates p73 and induces Bax-dependent apoptosis in cisplatin-resistant ovarian cancer cells

Several studies in the last years evidenced that deregulation of proapoptotic and antiapoptotic pathways are key players in the onset and maintenance of chemoresistance in advanced ovarian cancers. To characterize the signaling events and molecules involved in the acquisition of cisplatin resistance, we used the human ovarian cancer cell line A2780 and its derivative cisplatin-resistant subline A2780 CIS. We found that the mitochondrial intrinsic apoptotic pathway, induced by cis-dichlorodiammineplatinum (CDDP) in A2780 wild-type cells, was compromised in the resistant subline CIS. The analysis of expression of proteins involved in mitochondria-dependent apoptosis revealed a role of Bax and p73 but not p53. Indeed, we found that CDDP treatment induced the up-regulation of p53 in both sensitive and resistant A2780 cell lines. By contrast, p73 and Bax expressions were compromised in resistant cells. Pretreatment of resistant A2780 CIS cells with the histone deacetylase inhibitor trichostatin A overcomes apoptosis resistance to CDDP by restoring both p73 and Bax but not p53 expression. Altogether, these data indicate that p73, but not p53, is involved in the regulation of apoptosis susceptibility to cisplatin in A2780 ovarian cancer cells and evidence a key contribution of histone deacetylase activation in the acquisition of chemotherapy resistance in human ovarian cancer cells. [Mol Cancer Ther 2008;7(6):1410–9]

Looking at Drug Resistance Mechanisms for Microtubule Interacting Drugs: Does TUBB3 Work?

Vinca alkaloids and taxanes represent the mainstay of medical treatment of hematological and solid tumors. Unfortunately, a major clinical problem with these agents is drug resistance. Although a plethora of mechanisms of drug resistance have been described, only a few of them have been validated in clinical trials. Among these, the one involving the protein TUBB3 seems to represent a promising target for studying drug resistance. In fact, it seems that this protein is a factor promoting cell survival and represents an endogenous element of an inherent drug-resistance program built into cells to counteract the activity of microtubule-interacting drugs. Its pivotal role has been ascertained in clinical trials in lung, breast, and ovarian cancer, three diseases that can be successfully treated with microtubule-interacting drugs. Although TUBB3 is probably not a unique factor in drug resistance, the hope is that direct targeting of this protein will increase the response to microtubule-interacting drugs, thereby overcoming an important element in the growth of drug resistance.

Chemoresistant tumor cell lines display altered epidermal growth factor receptor and HER3 signaling and enhanced sensitivity to gefitinib

Deregulated signaling through the epidermal growth factor receptor (EGFR) is involved in chemoresistance. To identify the molecular determinants of sensitivity to the EGFR inhibitor gefitinib (Iressa, ZD1839) in chemoresistance, we compared the response of matched chemosensitive and chemoresistant glioma and ovarian cancer cell lines. We found that chemoresistant cell lines were 2‐ to 3‐fold more sensitive to gefitinib growth‐inhibitory effects, because of decreased proliferation rather than survival. Sensitivity to gefitinib correlated with overexpression and constitutive phosphorylation of HER2 and HER3, but not EGFR, altered HER ligand expression, and enhanced activation of EGF‐triggered EGFR pathway. No activating mutations were found in EGFR. Gefitinib fully inhibited EGF‐induced and constitutive Akt activation only in chemoresistant cells. In parallel, gefitinib downregulated constitutively phosphorylated HER2 and HER3, and activated GSK3β with a concomitant degradation of cyclin D1. Ectopically overexpressed HER2 on its own was insufficient to sensitize chemonaive cells to gefitinib. pHER3 coimmunoprecipitated with p85‐PI3K in chemoresistant cells and gefitinib dissociated these complexes. siRNA‐mediated inhibition of HER3 decreased constitutive activation of Akt and sensitivity to gefitinib in chemoresistant cells. Our study indicates that in chemoresistant cells gefitinib inhibits both an enhanced EGF‐triggered pathway and a constitutive HER3‐mediated Akt activation, indicating that inhibition of HER3 together with that of EGFR could be relevant in chemorefractory tumors. Furthermore, in combination experiments gefitinib enhanced the effects of coadministered drugs more in chemoresistant than chemosensitive ovarian cancer cells. Combined treatment might be therapeutically beneficial in chemoresistant tumors from ovary and likely from other tissues.

Bcl-2, Bax, Bcl-xL and Bcl-xS Expression in Neoplastic and Normal Endometrium

The two anti-apoptotic proteins Bcl-2 and Bcl-xL, and the two pro-apoptotic proteins Bax and Bcl-xS were measured by Western blotting in 51 neoplastic and 8 normal endometrial samples. The corresponding mRNA levels were analyzed by semiquantitative reverse transcriptase-polymerase chain reaction in a subgroup of 19 endometrial carcinomas. Neoplastic tissues had higher amounts of Bcl-2 protein than normal tissues (p < 0.051). Bcl-xL followed the same trend since its levels were higher in tumor than in normal samples (p < 0.048). Interestingly, Bcl-2 and Bcl-xL protein content showed a trend towards an inverse correlation (r = 0.27, p < 0.052). mRNA and protein levels directly correlated only with Bcl-2 (r = 0.63, p < 0.0032). Despite the fact that the amounts of Bcl-2, Bax and Bcl-xL proteins in the neoplastic population were not significantly differently distributed according to the clinicopathological features of the patients, the differences observed between normal and neoplastic samples suggest that these proteins may play a role in endometrial carcinoma: long-term follow-up studies will be required to confirm this hypothesis.