The mechanism of action of the oncogene bcl-2, a key regulator of the apoptotic process, is still debated. We have employed organelle-targeted chimeras of the Ca2+-sensitive photoprotein, aequorin, to investigate in detail the effect of Bcl-2 overexpression on intracellular Ca2+ homeostasis. In the ER and the Golgi apparatus, Bcl-2 overexpression increases the Ca2+ leak (while leaving Ca2+ accumulation unaffected), hence reducing the steady-state [Ca2+] levels. As a direct consequence, the [Ca2+] increases caused by inositol 1,4,5 trisphosphate (IP3)-generating agonists were reduced in amplitude in both the cytosol and the mitochondria. Bcl-2 overexpression also reduced the rate of Ca2+ influx activated by Ca2+ store depletion, possibly by an adaptive downregulation of this pathway. By interfering with Ca2+-dependent events at multiple intracellular sites, these effects of Bcl-2 on intracellular Ca2+ homeostasis may contribute to the protective role of this oncogene against programed cell death.

Although longstanding experimental evidence has associated alterations of calcium homeostasis to cell death, only in the past few years the role of calcium in the signaling of apoptosis has been extensively investigated. In this review, we will summarize the current knowledge, focusing on (i) the effect of the proteins of the Bcl-2 family on ER Ca 2 þ levels, (ii) the action of the proteolytic enzymes of apoptosis on the Ca 2 þ signaling machinery, (iii) the ensuing alterations on the signaling patterns of extracellular stimuli, and (iv) the intracellular targets of 'apoptotic' Ca 2 þ signals, with special emphasis on the mitochondria and cytosolic Ca 2 þ -dependent enzymes.

To better understand the functional role of the mitochondrial network in shaping the Ca 2؉ signals in living cells, we took advantage both of the newest genetically engineered green fluorescent protein-based Ca 2؉ sensors ("Cameleons," "Camgaroos," and "Pericams") and of the classical Ca 2؉ -sensitive photoprotein aequorin, all targeted to the mitochondrial matrix. The properties of the green fluorescent protein-based probes in terms of subcellular localization, photosensitivity, and Ca 2؉ affinity have been analyzed in detail. It is concluded that the ratiometric pericam is, at present, the most reliable mitochondrial Ca 2؉ probe for single cell studies, although this probe too is not devoid of problems. The results obtained with ratiometric pericam in single cells, combined with those obtained at the population level with aequorin, provide strong evidence demonstrating that the close vicinity of mitochondria to the Ca 2؉ release channels (and thus responsible for the fast uptake of Ca 2؉ by mitochondria upon receptor activation) are highly stable in time, suggesting the existence of specific interactions between mitochondria and the endoplasmic reticulum.

Although the importance of mitochondria in patho-physiology has become increasingly evident, it remains unclear whether these organelles play a role in Ca2+ handling by skeletal muscle. This undefined situation is mainly due to technical limitations in measuring Ca2+ transients reliably during the contraction–relaxation cycle. Using two-photon microscopy and genetically expressed “cameleon” Ca2+ sensors, we developed a robust system that enables the measurement of both cytoplasmic and mitochondrial Ca2+ transients in vivo. We show here for the first time that, in vivo and under highly physiological conditions, mitochondria in mammalian skeletal muscle take up Ca2+ during contraction induced by motor nerve stimulation and rapidly release it during relaxation. The mitochondrial Ca2+ increase is delayed by a few milliseconds compared with the cytosolic Ca2+ rise and occurs both during a single twitch and upon tetanic contraction.

We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK a (pK a ) around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca 2؉ ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca 2؉ accumulation by this organelle. Finally, we demonstrate that Ca 2؉ mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.

Skeletal muscle contraction depends on the release of Ca2+ from the sarcoplasmic reticulum (SR), but the dynamics of the SR free Ca2+ concentration ([Ca2+]SR), its modulation by physiological stimuli such as catecholamines, and the concomitant changes in cAMP handling have never been directly determined. We used two-photon microscopy imaging of GFP-based probes expressed in mouse skeletal muscles to monitor, for the first time in a live animal, the dynamics of [Ca2+]SR and cAMP. Our data, which were obtained in highly physiological conditions, suggest that free [Ca2+]SR decreases by ∼50 μM during single twitches elicited through nerve stimulation. We also demonstrate that cAMP levels rise upon β-adrenergic stimulation, leading to an increased efficacy of the Ca2+ release/reuptake cycle during motor nerve stimulation.

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Breast cancer (BC) is the most common cancer in women worldwide, and has an undeniable negative impact on public health. The advent of molecular biology and immunotherapy has made targeted therapeutic interventions possible, providing treatments tailored to the individual characteristics of the patient and the disease. The over-expression of human epidermal growth factor receptor (HER) 2 is implicated in the pathophysiology of BC and represents a clinically relevant biomarker for its treatment. Trastuzumab, a recombinant antibody targeting HER2, was the first biological drug approved for the treatment of HER2-positive BC. Although there are currently other anti-HER2 agents available (e.g. pertuzumab and lapatinib), trastuzumab remains the gold standard for treatment of this disease subtype. Nonetheless, concerns have been raised regarding potential cardiotoxicity and treatment resistance. Moreover, several other therapeutic issues remain unclear and have been addressed in an inconsistent way. The current literature lacks a comprehensive review of trastuzumab providing useful information for clinical practice, including pharmacokinetic and pharmacodynamic aspects, its clinical use, existing controversies and future advances. This detailed review of trastuzumab in the pharmacotherapy of BC attempts to fill this gap.