CMA mediates resistance in breast cancer models.

BACKGROUND: Breast cancer (BC) is the most common malignancy in women and the second leading cause of cancer-related death; chemoresistance is still a clinical challenge mainly because of the different molecular features of this kind of tumour. Doxorubicin (Doxo) is widely used despite its adverse effects and the common onset of resistance. Chaperone-Mediated Autophagy (CMA) has been identified as an important mechanism through which chemotherapeutics can exert their cytotoxic effects and, in this context, LAMP-2A, the key player of CMA, can be a useful biomarker. METHODS: A cohort of patients and breast cancer cells have been screened for Doxo effect and CMA activation by analysing the LAMP-2A level. Molecular silencing has been used to clarify CMA role in BC responsiveness to treatments. Low Doxo doses were combined with other drugs (TMZ or PX-478, a HIF-1α inhibitor) to evaluate their cytotoxic ability and their role in modulating CMA. RESULTS: In this paper, we showed that CMA is an important mechanism mediating the responsiveness of breast cancer cell to different treatments (Doxo and TMZ, as suggested by triple negative cells that are TMZ-resistant and fails to activate CMA). The LAMP-2A expression level was specific for different cell lines and patient-derived tumour subtypes, and was also useful in discriminating patients for their survival rates. Moreover, molecular silencing or pharmacological blockage of HIF-1α activity reverted BC resistance to TMZ. The combination of low-dose Doxo with TMZ or PX-478 showed that the drug associations have synergistic behaviours. CONCLUSION: Here, we demonstrated that CMA activity exerts a fundamental role in the responsiveness to different treatments, and LAMP-2A can be proposed as a reliable prognostic biomarker in breast cancer. In this context, HIF-1α, a potential target of CMA, can also be assessed as a valuable therapeutic target in BC in view of identifying new, more efficient and less toxic therapeutic drug combinations. Moreover, the possibility to combine Doxo with other drugs acting on different but coherent molecular targets could help overcome resistance and open the way to a decrease in the dose of the single drugs.

Diagnostic Circulating miRNAs in Sporadic Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the neurodegeneration of motoneurons. About 10% of ALS is hereditary and involves mutation in 25 different genes, while 90% of the cases are sporadic forms of ALS (sALS). The diagnosis of ALS includes the detection of early symptoms and, as disease progresses, muscle twitching and then atrophy spreads from hands to other parts of the body. The disease causes high disability and has a high mortality rate; moreover, the therapeutic approaches for the pathology are not effective. miRNAs are small non-coding RNAs, whose activity has a major impact on the expression levels of coding mRNA. The literature identifies several miRNAs with diagnostic abilities on sALS, but a unique diagnostic profile is not defined. As miRNAs could be secreted, the identification of specific blood miRNAs with diagnostic ability for sALS could be helpful in the identification of the patients. In the view of personalized medicine, we performed a meta-analysis of the literature in order to select specific circulating miRNAs with diagnostic properties and, by bioinformatics approaches, we identified a panel of 10 miRNAs (miR-193b, miR-3911, miR-139-5p, miR-193b-1, miR-338-5p, miR-3911-1, miR-455-3p, miR-4687-5p, miR-4745-5p, and miR-4763-3p) able to classify sALS patients by blood analysis. Among them, the analysis of expression levels of the couple of blood miR-193b/miR-4745-5p could be translated in clinical practice for the diagnosis of sALS.

A mutation in a novel yeast proteasomal gene, RPN11/MPR1, produces a cell cycle arrest, overreplication of nuclear and mitochondrial DNA, and an altered mitochondrial morphology.

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24 degreesC growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36 degreesC on either carbon source. Microscopic observation of cells growing on glucose at 24 degreesC shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho degrees] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Towards a blueprint of the cell cycle.

The understanding of the organisation of cell cycle events is of utmost importance to devise effective therapeutic strategies for cancer. In this article we gather evidences from the literature in support of a system model of the cell cycle, in which a growth-sensitive threshold controls entry into S phase and the sequential activation of cyclin-dependent kinases. The cycle is terminated by an End function, that comprises events from the onset of mitosis to cell division and that may also be modulated by the increase of cell size. This blueprint allows quantitative predictions by computer simulations of steady and transitory states. In fact, we show that the proposed control system applies to budding yeast populations during nutritional shift-up and following hyperactivation of the cAMP signalling pathway. Besides the growth-sensitive control system it is shown to apply to mammalian cells both in the exit from quiescence and in active proliferation. The putative molecular determinants that set the threshold controlling S phase entry are consistently altered in cancer cells. Finally, we discuss an input/output analysis based on the simulated behaviour derived from the blueprint as a new tool to investigate the road to cancer.

Opposite effects of TPA on G1/S transition and on cell size in the low metastatic B16F1 with respect to high metastatic BL6 murine melanoma cells.

Phorbol esters, known activators of c- and n-protein kinase C (PKC) isoforms, play a pivotal role in tumor promotion. In order to better understand the relationships between PKC activation, the metastatic potential and the proliferative capacity, we have analyzed the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment on the proliferative as well as on the cell cycle distribution and on the cell size of low and high metastatic murine B16F1 and B16-BL6 (BL6) melanoma cells, respectively. TPA-treated B16F1 cells showed an increased proliferative capacity up to 72 h, the cytofluorimetric analysis revealing an increased number of cells in the S + G2-M phase of the cell cycle. In contrast, TPA-treated BL6 cells reached a plateau at 48 h and showed an increased cell volume in the G1 and S phases of the cell cycle, with a reduction in the percentage of cells in the S + G2-M phase. Taken together, our results indicate opposite effects of TPA treatment in murine melanoma cells of different metastatic potential. TPA could cause a block in the G1 phase of the cell cycle with enhanced cell volume in high metastatic BL6 cells. The same treatment, on the contrary, induced an increased entry into the cell cycle of low metastatic B16F1 cells, suggesting a relationship between cell proliferation and the metastatic potential of B16 murine melanoma cells. Moreover, under the present conditions, classical PKC isoforms were inactivated, suggesting the involvement of the TPA-dependent novel PKCs.

Development of a pH-controlled fed-batch system for budding yeast.

In the manufacturing of baker's yeast by aerobic fed-batch systems, continuous assessment of the state of the process is necessary for regulating the flow rate (on/off) for growth medium addition. A new, simple method for the fed-batch yeast process has been developed. It is based on pH changes as a suitable parameter for regulating the feed of fresh concentrated medium in response to metabolic activities of the yeast population. Experimental results have shown that it enables the attaining of high cell density with both high productivity and high yields.

Identification of different daughter and parent subpopulations in an asynchronously growing Saccharomyces cerevisiae population.

Under all growth conditions, a growing Saccharomyces cerevisiae yeast population is extremely heterogeneous, since individual cells differ in their cell size; this is due to their position in the cell division cycle and their genealogical age. To gain insight into the structure of a growing yeast population, we used a recently developed flow cytometric approach which enables, in asynchronously growing S. cerevisiae populations, tagging of both the cell age and the protein content of individual cells. This approach enabled the identification of daughter cells belonging to different cell cycle positions (i.e. newborn, G1, S + G2 + M + G1*, and dividing), thus yielding information about the relative fraction in the whole population, cell size and variability. More limited information could be obtained for the parent subpopulation; however, we were able to identify and characterize the dividing parent cells. The coefficient of variation (CV) of the protein content distribution for the dividing parents (27) was much higher than the CV of dividing daughters (18). Further findings obtained indicated a large overlap between the cell protein content distributions of daughter and parent cells as well as between the protein content of cells of the same subpopulation but belonging to different stages of the cell division cycle. The analysis of these differences enables a better understanding of the complex structure of an asynchronously growing yeast population.

Relation between growth dynamics and diffusional limitations in Saccharomyces cerevisiae cells growing as entrapped in an insolubilised gelatin gel.

Flow-cytometric analysis was employed to investigate growth dynamics of a yeast cell population immobilised in an insolubilised gelatin gel by means of the quantitative determination of the average protein content per cell. This analysis was carried out on both the immobilised cell population considered as a whole and the subpopulations colonising the gelatin matrix at different depths. The results show that growth of the gelatin-immobilised yeast population was affected by the existence of a gradient of nutrient concentrations through the matrix and are in agreement with the unsteady-state diffusion model employed for the description of glucose transfer in the gel.

NADH reoxidation does not control glycolytic flux during exposure of respiring Saccharomyces cerevisiae cultures to glucose excess.

Introduction of the Lactobacillus casei lactate dehydrogenase (LDH) gene into Saccharomyces cerevisiae under the control of the TPI1 promoter yielded high LDH levels in batch and chemostat cultures. LDH expression did not affect the dilution rate above which respiro-fermentative metabolism occurred (Dc) in aerobic, glucose-limited chemostats. Above Dc, the LDH-expressing strain produced both ethanol and lactate, but its overall fermentation rate was the same as in wild-type cultures. Exposure of respiring, LDH-expressing cultures to glucose excess triggered simultaneous ethanol and lactate production. However, the specific glucose consumption rate was not affected, indicating that NADH reoxidation does not control glycolytic flux under these conditions.

Relating growth dynamics and glucoamylase excretion of individual Saccharomyces cerevisiae cells.

We have developed a novel flow cytometric procedure that allows determinations of properties of protein excretion in the growth medium on a cell-by-cell basis in Saccharomyces cerevisiae. The procedure is based on labelling of a periplasmically secreted protein with antibodies conjugated to a fluorescent marker such as fluorescein isothiocyanate (FITC). The staining conditions did not perturb cell growth after resuspension of stained cells in growth medium. Decrease in fluorescence was found to correlate with excretion of glucoamylase into the growth medium. The analysis of the staining pattern over time provides information on the behaviour of individual cells belonging to different cell-cycle phases and can be used to calculate the specific excretion rate of the overall population.

Real-time flow cytometric quantification of GFP expression and Gfp-fluorescence generation in Saccharomyces cerevisiae.

A genetic and analytical methodology was developed based on a green fluorescent mutant protein (Gfp(S65T)) that allows the real-time quantification of gene expression in Saccharomyces cerevisiae. Using the UAS(GAL)(1-10)/CYC1 promoter and plasmids that are maintained in different copy numbers per cell, wild-type GFP and mutant GFP(S65T) were expressed in low to high concentration. Flow cytometric analysis was then applied to directly quantify Gfp((S65T)) (both wild type and mutant protein) expression at the single-cell level, and to indirectly measure the concentrations of non-fluorescent apoGfp((S65T)) and fluorescent Gfp((S65T)), which is autocatalytically formed from the apoprotein. Kinetics of apoGfp((S65T))/Gfp((S65T)) conversion during aerobic growth showed that the time required for complete apoGfp((S65T)) conversion is limited only by the amount of apoprotein that is expressed. When GFP(S65T) was expressed in single copy, the apoprotein did not accumulate and was instantly converted into its fluorescent form. The data indicate that an instant quantification of gene expression in S. cerevisiae is achievable based on Gfp(S65T), even if the gene is transcribed from a very strong promoter.

Higher levels of melanin and inhibition of cdk2 activity in primary human melanoma cells WM115 overexpressing nPKCdelta.

Many studies have attempted to define the state of differentiation of melanoma cells and to correlate it with other critical parameters of malignancy such as the tumorigenic and metastatic nature of the cells. In the present paper we focused on the possible relationships between the novel protein kinase C isoform nPKCdelta, melanin synthesis and proliferative capacity in a primary human melanoma cell line WM115. Cells were transfected to produce overexpression of this isoform and the effects on melanin synthesis, cyclin-E dependent kinase (cdk2) activity and cyclin E expression were studied. It was shown that translocation of nPKCdelta into the nucleus affects melanin synthesis and inhibits cdk2 activity. As a compensatory effect, the level of cyclin E increases. In view of these results we suggest a model for the role of nPKCdelta in melanoma cells that may offer a new therapeutic perspective.

Overexpression of novel protein kinase C delta in BL6 murine melanoma cells inhibits the proliferative capacity in vitro but enhances the metastatic potential in vivo.

In this study we analysed the effect of overexpressing novel protein kinase C delta isoform (n-PKC delta) on melanin synthesis and metastatic potential in the highly metastatic BL6 murine melanoma cells. The proliferative capacity in vitro and into matrigel in vivo were also examined. Although murine melanocytes express the n-PKC delta isoform, BL6 cells do not express this isoform at levels detectable by Western blot analysis. In untransfected and transfected cells we also studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a modulator of specific isoforms of PKC, and of bryostatin 1, a potent immunomodulator and antineoplastic drug and a partial agonist of PKC. Our results demonstrate a pivotal role for this isoform in melanin synthesis and the close relationship between n-PKC delta expression and its association with the particulate fraction, melanogenesis and metastatic potential. In fact, heterogeneous BL6 cells overexpressing n-PKC delta and all the clones isolated showed increased intracellular melanin and metastatic capacity. TPA and bryostatin 1 decreased n-PKC delta expression, the intracellular melanin level and metastatic capacity in both cell lines. Therefore both treatments were able to abolish the effects of overexpressing n-PKC delta.

Tracking of individual cell cohorts in asynchronous Saccharomyces cerevisiae populations.

A novel flow cytometric procedure has been developed with the aim to obtain the growth properties of individual Saccharomyces cerevisiae cells in asynchronous culture. The method is based on labeling of the cell surface with FITC-conjugated concanavalin A and detection of the single-cell fluorescence with flow cytometry after cell exposure to growth conditions. Because the formation of new cell wall material in budded cells is restricted to the bud tip, exposure of the stained cells to growth conditions results in three cell types: (i) stained cells, (ii) partially stained cells, and (iii) unstained cells. Analysis of the staining pattern over time permits the determination of the specific growth rate of the cell population, the length of the budded cell cycle phase, and the growth pattern during the cell cycle of newly formed, partially stained daughter cells. The procedure has been tested with yeast cell populations growing at different rates. The data suggest an exponential increase in the size of individual cells during the cell cycle, as reflected by the forward angle light scattering (FALS) signals. It has been found that the apparent single-cell specific cell size growth rates, determined by FALS intensity, are significantly lower than the specific growth rates of the overall population. This could indicate that the tracking of a cohort of cells is significantly perturbed by a distribution of staining levels of daughter cells at cell division and that FALS may not be a good indicator of the cell size.

Flow cytometry and cell cycle kinetics in continuous and fed-batch fermentations of budding yeast.

Cell size distributions, obtained either as protein distribution by flow cytometry or as cell volume distribution by a Coulter counter, give relevant information about the growth conditions of populations of budding yeast Saccharomyces cerevisiae. We have previously found a good correlation between these distributions and the growth rate in continuous cultures (Ranzi et al., Biotechnol. Bioeng. 1986, 28, 185-190). We now present determinations of the protein distributions and cell volume distributions during different fed-batch fermentations performed with a simple on/off controller. Since during the fed-batch fermentation a true steady state is not obtained, the distributions continuously change with time, but nevertheless we observed a good correlation between the average of both distributions and the actual growth rate. The behavior of the cell size distributions can be interpreted on the basis of a two-threshold cell cycle model in which both the critical protein content at budding (Ps) and the critical protein content for cell division (Pm) are differently modulated by the growth rate. Additional findings will be presented showing that this model can be used to successfully explain the insurgence and the maintenance of oscillatory states in continuous cultures.

Development of metabolically engineered Saccharomyces cerevisiae cells for the production of lactic acid.

Interesting challenges from metabolically engineered Saccharomyces cerevisiae cells arise from the opportunity to obtain yeast strains useful for the production of chemical(s). In this paper, we describe the accumulation of lactic acid in the culture medium of growing, engineered yeast cells expressing a mammalian lactate dehydrogenase gene (LDH-A). High and reproducible productions (20 g/L) and productivities (up to 11 g/L/h) of lactic acid have been obtained by modulating the physiological growth conditions. Since yeast cells are acid tolerant and survive at very low pH values, the production of lactate can be avoided. In perspective, the approaches described could be useful for the production of lactic acid, outflanking the problems related to the synthesis from bacteria cells. In fact, during industrial productions, there is an inhibitory effect on the metabolic activities of the growing bacteria (i.e., Lactobacillus spp.) caused by the acid produced and by the low pH value. Thus, strategies to prevent the lowering of pH are conventional operations. These processes allow the production of lactate(s) and require the purification of the acid from its salt. The biotechnological implications of this study are also discussed.

Heterologous gene expression in continuous cultures of budding yeast.

In a previous paper we have studied the expression of beta-galactosidase from Escherichia coli, driven from the inducible GAL1-10/CYC1 hybrid promoter, in batch cultures of budding Saccharomyces cerevisiae and have described operating conditions for maximal productivity. In this paper we show that the plasmid instability in continuous cultures can be overcome by utilizing appropriate selection markers and a high copy number vector. The maximal level of expression is influenced by the dilution rate. Moreover, enzyme accumulation appears to depend also upon the degree of oxygenation. A possible explanation of these modulations is discussed, taking into account the interactions of the UAS-GAL and TATA-CYC1 elements.

Oscillations in continuous cultures of budding yeast: a segregated parameter analysis.

Sustained oscillations have been observed in continuous cultures of Saccharomyces cerevisiae. These oscillations appear spontaneously under aerobic conditions and may constitute a severe limitation for process control. We have found that oscillations arise only in a well defined range of dilution rates and dissolved oxygen values. The period of the oscillations is related, but not equal, to the mass doubling time, and shows a relation ship with both the parent cells and daughter cells generation times. At high dilution rates two oscillatory regimens, with different periods, are observed. The analysis of the budding index shows a marked degree of synchronization of the culture, however significant differences, both in phase and in amplitude, are ob served if the budding index of parent cells and of daughter cells are considered separately. The complex changes of the cell population are clearly demonstrated by the continuous and periodic modification of both cell volume distributions and protein distributions. Ethanol is always accumulated before the drop of dissolved oxygen concentration and one of the peaks of budding index. We propose a model that explains the insurgence of these oscillation as a consequence of changes in cell cycle parameters due to alternate growth in glucose and in ethanol.

Involvement of the alpha isoenzyme of protein kinase C in the growth inhibition induced by phorbol esters in MH1C1 hepatoma cells.

MH1C1 rat hepatoma cells express the alpha isoenzyme as the only phorbol-ester sensitive isoform of protein kinase C (PKC). In this cell line, phorbol 12-myristate 13-acetate (PMA) induced a marked, dose-dependent growth inhibition. The administration of the PKC inhibitor staurosporine was able to mimic the effect of the phorbol ester on cell growth in a dose-dependent fashion, whereas the PKC activator arachidonic acid stimulated cell proliferation. Exposure of cells to an antisense oligonucleotide specific for alpha PKC caused a significant impairment of cell growth. These data suggest that the alpha PKC activity is required for proliferation of MH1C1 cells.

Involvement of a cell size control mechanism in the induction and maintenance of oscillations in continuous cultures of budding yeast.

Spontaneous oscillations occur in glucose-limited continuous cultures of Saccharomyces cerevisiae under aerobic conditions. The oscillatory behavior is detectable as a periodic change of many bioparameters such as dissolved oxygen, ethanol production, biomass concentration, as well as cellular content of storage carbohydrates and is associated to a marked synchronization of the yeast population. These oscillations may be related to a periodic accumulation of ethanol produced by yeast in the culture medium.The addition of ethanol to oscillating yeast cultures supports this hypothesis: indeed, no effect was observed if ethanol was added when already present in the medium, while a marked phase oscillation shift was obtained when ethanol was added at any other time. Moreover, the addition of ethanol to a nonoscillating culture triggers new oscillations. An accurate analysis performed at the level of nonoscillating yeast populations perturbed by addition of ethanol showed that both the growth rate and the protein content required for cell division increased in the presence of mixed substrate (i.e., ethanol plus limiting glucose). A marked synchronization of the yeast population occurred when the added ethanol was exhausted and the culture resumed growth only on limiting glucose. A decrease of protein content required for cell division was also apparent. These experimental findings support a new model for spontaneous oscillations in yeast cultures in which the alternative growth on limiting glucose and limiting glucose plus ethanol modifies the critical protein content required for cell division.