Stanislaw Krajewski

Apoptosis in the Failing Human Heart

BACKGROUND Loss of myocytes is an important mechanism in the development of cardiac failure of either ischemic or nonischemic origin. However, whether programmed cell death (apoptosis) is implicated in the terminal stages of heart failure is not known. We therefore studied the magnitude of myocyte apoptosis in patients with intractable congestive heart failure. METHODS Myocardial samples were obtained from the hearts of 36 patients who underwent cardiac transplantation and from the hearts of 3 patients who died soon after myocardial infarction. Samples from 11 normal hearts were used as controls. Apoptosis was evaluated histochemically, biochemically, and by a combination of histochemical analysis and confocal microscopy. The expression of two proto-oncogenes that influence apoptosis, BCL2 and BAX, was also determined. RESULTS Heart failure was characterized morphologically by a 232-fold increase in myocyte apoptosis and biochemically by DNA laddering (an indicator of apoptosis). The histochemical demonstration of DNA-strand breaks in myocyte nuclei was coupled with the documentation of chromatin condensation and fragmentation by confocal microscopy. All these findings reflect apoptosis of myocytes. The percentage of myocytes labeled with BCL2 (which protects cells against apoptosis) was 1.8 times as high in the hearts of patients with cardiac failure as in the normal hearts, whereas labeling with BAX (which promotes apoptosis) remained constant. The near doubling of the expression of BCL2 in the cardiac tissue of patients with heart failure was confirmed by Western blotting. CONCLUSIONS Programmed death of myocytes occurs in the decompensated human heart in spite of the enhanced expression of BCL2; this phenomenon may contribute to the progression of cardiac dysfunction.

Cloning and functional analysis of BAG-1: A novel Bcl-2-binding protein with anti-cell death activity

Using a protein interaction cloning technique, we identified cDNAs that encode a novel Bcl-2-binding protein, termed BAG-1. The BAG-1 protein shares no significant homology with Bcl-2 or other Bcl-2 family proteins, which can form homo- and heterodimers. In gene transfer experiments using a human lymphoid cell line, Jurkat, coexpression of BAG-1 and Bcl-2 provided markedly increased protection from cell death induced by several stimuli, including staurosporine, anti-Fas antibody, and cytolytic T cells, relative to cells that contained gene transfer-mediated elevations in either BAG-1 or Bcl-2 protein alone. BAG-transfected 3T3 fibroblasts also exhibited prolonged cell survival in response to an apoptotic stimulus. The findings indicate that bag-1 represents a new type of anti-cell death gene and suggest that some routes of apoptosis induction previously ascribed to Bcl-2-independent pathways may instead reflect a need for the combination of Bcl-2 and BAG-1.

BCL‐2 family proteins: Regulators of cell death involved in the pathogenesis of cancer and resistance to therapy

The BCL‐2 gene was first discovered because of its involvement in the t(14;18) chromosomal translocations commonly found in lymphomas, which result in deregulation of BCL‐2 gene expression and cause inappropriately high levels of Bcl‐2 protein production. Expression of the BCL‐2 gene can also become altered in human cancers through other mechanisms, including loss of the p53 tumor suppressor which normally functions as a repressor of BCL‐2 gene expression in some tissues. Bcl‐2 is a blocker of programmed cell death and apoptosis that contributes to neoplastic cell expansion by preventing cell turnover caused by physiological cell death mechanisms, as opposed to accelerating rates of cell division. Overproduction of the Bcl‐2 protein also prevents cell death induced by nearly all cytotoxic anticancer drugs and radiation, thus contributing to treatment failures in patients with some types of cancer. Several homologs of Bcl‐2 have recently been discovered, some of which function as inhibitors of cell death and others as promoters of apoptosis that oppose the actions of the Bcl‐2 protein. Many of these Bcl‐2 family proteins can interact through formation of homo‐ and heterotypic dimers. In addition, several nonhomologous proteins have been identified that bind to Bcl‐2 and that can modulate apoptosis. These protein‐protein interactions may eventual serve as targets for pharmacologically manipulating the physiological cell death pathway for treatment of cancer and several other diseases.

Targeting the prostate for destruction through a vascular address

Organ specific drug targeting was explored in mice as a possible alternative to surgery to treat prostate diseases. Peptides that specifically recognize the vasculature in the prostate were identified from phage-displayed peptide libraries by selecting for phage capable of homing into the prostate after an i.v. injection. One of the phage selected in this manner homed to the prostate 10–15 times more than to other organs. Unselected phage did not show this preference. The phage bound also to vasculature in the human prostate. The peptide displayed by the prostate-homing phage, SMSIARL (single letter code), was synthesized and shown to inhibit the homing of the phage when co-injected into mice with the phage. Systemic treatment of mice with a chimeric peptide consisting of the SMSIARL homing peptide, linked to a proapoptotic peptide that disrupts mitochondrial membranes, caused tissue destruction in the prostate, but not in other organs. The chimeric peptide delayed the development of the cancers in prostate cancer-prone transgenic mice (TRAMP mice). These results suggest that it may be possible to develop an alternative to surgical prostate resection and that such a treatment may also reduce future cancer risk.

Myocardial Cell Death in Fibrillating and Dilated Human Right Atria

OBJECTIVES The aim of the present study was to determine if myocytes can die by apoptosis in fibrillating and dilated human atria. BACKGROUND The cellular remodeling that occurs during atrial fibrillation (AF) may reflect a degree of dedifferentiation of the atrial myocardium, a process that may be reversible. METHODS We examined human right atrial myocardium specimens (n = 50) for the presence of apoptotic myocytes. We used immunohistochemical and Western blotting analysis to examine the expression of a final effector of programmed cell death, caspase-3 (CASP-3) and of regulatory proteins from the BCL-2 family. RESULTS Sections from atria in AF contained a high percentage of large myocytes with a disrupted sarcomeric apparatus replaced by glycogen granules (64.4 +/- 6.3% vs. 12.2 +/- 5.8%). These abnormal myocytes, which also predominated in atria from hearts with decreased left ventricular ejection fraction (42.3 +/- 10.1%), contained large nuclei, most of which were TUNEL positive, indicating a degree of DNA breakage. None of these abnormal myocytes expressed the proliferative antigen Ki-67. A small percentage of the enlarged nuclei (4.2 +/- 0.8%) contained condensed chromatin and were strongly TUNEL positive. Both the pro- and activated forms of CASP-3 were detected in diseased myocardial samples, which also showed stronger CASP-3 expression than controls. Expression of the antiapoptotic BCL-2 protein was decreased in diseased atria, whereas that of the proapoptotic BAX protein remained unchanged. CONCLUSIONS In fibrillating and dilated atria, apoptotic death of myocytes with myolysis contributes to cellular remodeling, which may not be entirely reversible.

Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease

AbstractHuntingtons disease (HD) is an autosomal dominant progressive neurodegenerative disorder resulting in selective neuronal loss and dysfunction in the striatum and cortex. The molecular pathways leading to the selectivity of neuronal cell death in HD are poorly understood. Proteolytic processing of full-length mutant huntingtin (Htt) and subsequent events may play an important role in the selective neuronal cell death found in this disease. Despite the identification of Htt as a substrate for caspases, it is not known which caspase(s) cleaves Htt in vivo or whether regional expression of caspases contribute to selective neuronal cells loss. Here, we evaluate whether specific caspases are involved in cell death induced by mutant Htt and if this correlates with our recent finding that Htt is cleaved in vivo at the caspase consensus site 552. We find that caspase-2 cleaves Htt selectively at amino acid 552. Further, Htt recruits caspase-2 into an apoptosome-like complex. Binding of caspase-2 to Htt is polyglutamine repeat-length dependent, and therefore may serve as a critical initiation step in HD cell death. This hypothesis is supported by the requirement of caspase-2 for the death of mouse primary striatal cells derived from HD transgenic mice expressing full-length Htt (YAC72). Expression of catalytically inactive (dominant-negative) forms of caspase-2, caspase-7, and to some extent caspase-6, reduced the cell death of YAC72 primary striatal cells, while the catalytically inactive forms of caspase-3, -8, and -9 did not. Histological analysis of post-mortem human brain tissue and YAC72 mice revealed activation of caspases and enhanced caspase-2 immunoreactivity in medium spiny neurons of the striatum and the cortical projection neurons when compared to controls. Further, upregulation of caspase-2 correlates directly with decreased levels of brain-derived neurotrophic factor in the cortex and striatum of 3-month YAC72 transgenic mice and therefore suggests that these changes are early events in HD pathogenesis. These data support the involvement of caspase-2 in the selective neuronal cell death associated with HD in the striatum and cortex.

Up-regulation of Bax Protein in Degenerating Retinal Ganglion Cells Precedes Apoptotic Cell Death after Optic Nerve Lesion in the Rat

Retrograde degeneration of retinal ganglion cells as a consequence of optic nerve lesion has been shown to fulfil the criteria of apoptosis. In the present study, we investigated the time course of ganglion cell apoptosis following intraorbital crushing of the optic nerve in adult rats using morphological criteria and applying a terminal transferase technique (TUNEL) for in situ detection of DNA strand breaks. In addition, we examined expression patterns of the anti‐apoptotic proteins Bcl‐2 and Bcl‐X and the cell death‐promoting protein Bax in retinae after crushing the optic nerve. Apoptotic nuclei were detected in the ganglion cell layer in the first 3 weeks after optic nerve crush, with a peak after 6 days. Bcl‐2 and Bcl‐X proteins were expressed in ganglion cells at low levels. Expression of Bcl‐2 decreased further during the days following crush. Bcl‐X expression was initially increased, followed by a decline over the following days. In contrast, Bax protein, which was expressed in most ganglion cells at moderate baseline levels, was sharply increased as early as 30 min after crush, reached peak levels after 3 days, and remained up‐regulated for at least 1 week thereafter. Double labelling for Bax and TUNEL in retinal sections, however, did not reveal colocalization of the two signals in individual retinal ganglion cells, consistent with the idea that increases in Bax precede apoptosis after optic nerve lesion. Thus, retinal ganglion cell death might be prevented by ablation of Bax protein in these cells, or by up‐regulation of Bax‐antagonists such as Bcl‐2 or Bcl‐X.

Bax is increased in the retina of diabetic subjects and is associated with pericyte apoptosis in vivo and in vitro.

Diabetes of even short duration accelerates the death of capillary cells and neurons in the inner retina by a process consistent with apoptosis. We examined whether the process is accompanied by changes in the expression of endogenous regulators of apoptosis. In postmortem retinas of 18 diabetic donors (age 67 +/- 6 years, diabetes duration 9 +/- 4 years) the levels of pro-apoptotic Bax were slightly, but significantly, increased when compared with levels in 20 age-matched nondiabetic donors (P = 0.04). In both groups, Bax localized to vascular and neural cells of the inner retina. Neither pro-apoptotic Bcl-X(S), nor pro-survival Bcl-X(L) appeared affected by diabetes. The levels of these molecules could not be accurately quantitated in lysates of retinal vessels because of variable degrees of glial contamination. However, studies in situ showed in several pericytes, the outer cells of retinal capillaries, intense Bax staining often in conjunction with DNA fragmentation. Bovine retinal pericytes exposed in vitro to high glucose levels for 5 weeks showed elevated levels of Bax (P = 0.03) and increased frequency of annexin V binding, indicative of early apoptosis. Hence, human diabetes selectively alters the expression of Bax in the retina and retinal vascular pericytes at the same time as it causes increased rates of apoptosis. The identical program induced by high glucose in vitro implicates hyperglycemia as a causative factor in vivo, and provides a model for establishing the role of Bax in the accelerated death of retinal cells induced by diabetes.

PREDICTING CHEMORESISTANCE IN HUMAN MALIGNANT GLIOMA CELLS: THE ROLE OF MOLECULAR GENETIC ANALYSES

Less than 30% of malignant gliomas respond to adjuvant chemotherapy. Here, we asked whether alterations in the p53 and RB pathways and the expression of six BCL‐2 family proteins predicted acute cytotoxicity and clonogenic cell death induced by BCNU, vincristine, cytarabine, teniposide, doxorubicin, camptothecin or β‐lapachone in 12 human malignant glioma cell lines. Neither wild‐type p53 status, nor p53 protein accumulation, nor p21 or MDM‐2 levels, nor differential expression of BCL‐2 family proteins predicted drug sensitivity, except for an association of BAX with higher β‐lapachone sensitivity in acute cytotoxicity assays. p16 protein expression was associated with high doubling time and chemoresistance. We conclude that some important molecular changes, which are involved in the development of gliomas and attributed a role in regulating vulnerability to apoptosis, may not determine the response to chemotherapy in these tumors. Int. J. Cancer (Pred. Oncol.) 79:640–644, 1998.

Synthetic triterpenoids cooperate with tumor necrosis factor-related apoptosis-inducing ligand to induce apoptosis of breast cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) has been shown to induce apoptosis specifically in cancer cells while sparing normal tissues. Unfortunately not all cancer cells respond to TRAIL; therefore, TRAIL sensitizing agents are currently being explored. We have identified synthetic triterpenoids, including 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivative 1-(2-cyano-3,12-dioxooleana-1,9-dien-28-oyl) imidazole (CDDO-Im), which sensitize TRAIL-resistant cancer cells to TRAIL-mediated apoptosis. Here we show that TRAIL-treated T47D and MDA-MB-468 breast cancer cells fail to initiate detectable caspase-8 processing and, consequently, do not initiate TRAIL-mediated apoptosis. Concomitant treatment with CDDO or CDDO-Im reverses the TRAIL-resistant phenotype, promoting robust caspase-8 processing and induction of TRAIL-mediated apoptosis in vitro. The combination of triterpenoids and monoclonal anti-TRAIL receptor-1 (DR4) antibody also induces apoptosis of breast cancer cells in vitro. From a mechanistic standpoint, we show that CDDO and CDDO-Im down-regulate the antiapoptotic protein c-FLIP(L), and up-regulate cell surface TRAIL receptors DR4 and DR5. CDDO and CDDO-Im, when used in combination with TRAIL, have no adverse affect on cultured normal human mammary epithelial cells. Moreover, CDDO-Im and TRAIL are well tolerated in mice and the combination of CDDO-Im and TRAIL reduces tumor burden in vivo in an MDA-MB-468 tumor xenograft model. These data suggest that CDDO and CDDO-Im may be useful for selectively reversing the TRAIL-resistant phenotype in cancer but not normal cells.