In vitro effect of different mediators of apoptosis on canine cranial and caudal cruciate ligament fibroblasts and its reversibility by pancaspase inhibitor zVAD.fmk

Primary fibroblast cultures of canine cranial (CCL) and caudal (CaCL) cruciate ligaments were stimulated with different apoptosis inducers with or without preincubation of the pancaspase inhibitor zVAD.fmk. In contrast to CaCL fibroblasts, fibroblasts from CCL were significantly more susceptible to apoptosis inducers of the intrinsic pathway like doxorubicin, cisplatin and nitric oxide (NO)-donors and to Fas ligand (FasL), an apoptosis inducer of the death receptor pathway. Apoptotic response to staurosporine and the peroxynitrite donor GEA was similar in both ligament fibroblasts. Stimulation with dexamethasone or TNFalpha could not induce apoptosis in CCL and CaCL fibroblasts, in spite of present TNFR1 and TNFR2 receptors. zVAD.fmk was able to prevent apoptosis in up to 66% of CCL cells when treated with FasL, cisplatin or doxorubicin but it had no effect on NO or peroxynitrite induced apoptosis. In conclusion, differential susceptibility to apoptotic triggers like FasL or NO between cranial and caudal cruciate ligament fibroblasts in vitro may be a reflection of the different susceptibilities to degenerative rupture of the ligament. These findings indicate that a general caspase inhibition does not completely protect canine CCL cells from apoptosis.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

Cytotoxic effects of camptothecin and cisplatin combined with tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in a model of primary culture of non-small cell lung cancer

The cytokine tumor-necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been shown to preferentially induce apoptosis in cancer cells. A previous study of our group demonstrated that non-small cell lung cancer cell lines can be sensitized to Apo2L/TRAIL-induced apoptosis by chemotherapeutic agents. The aim of the present study was the evaluation of these results in a model of primary culture of non-small cell lung cancer.

Rescue from apoptosis in early (CD34 selected) versus late (CD34 unselected) human hematopoietic cells by VLA-4 and VCAM-1 dependent adhesion to bone marrow stromal cells

The interaction of hematopoietic precursor cell with bone marrow stromal cells is assumed to be important to the survival of hematopoietic precursor cells during hematopoietic cell long-term culture. Early hematopoietic stem cells are preferentially found within the stromal adherent cell fraction in primary long-term bone marrow cultures. The purpose of this dissertation was to understand the molecular mechanisms that govern these interactions for the regulation of survival and proliferation of early versus late hematopoietic cells.^ Monoclonal antibodies to the VLA-4 recognize the alpha4 beta1 integrin receptor on human hematopoietic cells. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34 selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34 selected cells was shown to induce apoptosis of CD34 selected cells in these CD34 selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34 selected. Since there is no difference between the alpha4 beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+ selected precursor cells proliferate at a higher rate when these cells are plated on recombinant VCAM-1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function which results from the anchorage-dependent growth of the CD34 selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage-independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy. ^

Mechanisms of liposomal all-{\it trans\/} retinoic acid and vitamin D3 induced inhibition of cell proliferation and stimulation of apoptosis in aggressive B-cell non-Hodgkin's lymphoma

The Non-Hodgkin's Lymphoma (NHLs) are neoplasms of the immune system. Currently, less than 1% of the etiology of the 22,000 newly diagnosed lymphoma cases in the U.S.A. every year is known. This disease has a significant prevalence and high mortality rate. Cell growth in lymphomas has been shown to be an important parameter in aggressive NHL when establishing prognosis, as well as an integral part in the pathophysiology of the disease process. While many aggressive B cell NHLs respond initially to chemotherapeutic regimens such as CHOP-bleo (adriamycin, vincristine and bleomycin) etc., relapse is common, and the patient is then often refractory to further salvage treatment regimens.^ To assess their potential to inhibit aggressive B cell NHLs and induce apoptosis (also referred to as programmed cell death (PCD)), it was proposed to utilize the following biological agents-liposomal all-trans retinoic acid (L-ATRA) which is a derivative of Vitamin A in liposomes and Vitamin D3. Preliminary evidence indicates that L-ATRA may inhibit cell growth in these cells and may induce PCD as well. Detailed studies were performed to understand the above phenomena by L-ATRA and Vitamin D3 in recently established NHL-B cell lines and primary cell cultures. The gene regulation involved in the case of L-ATRA was also delineated. ^

Thiazolides promote apoptosis in colorectal tumor cells via MAP kinase-induced Bim and Puma activation

While many anticancer therapies aim to target the death of tumor cells, sophisticated resistance mechanisms in the tumor cells prevent cell death induction. In particular enzymes of the glutathion-S-transferase (GST) family represent a well-known detoxification mechanism, which limit the effect of chemotherapeutic drugs in tumor cells. Specifically, GST of the class P1 (GSTP1-1) is overexpressed in colorectal tumor cells and renders them resistant to various drugs. Thus, GSTP1-1 has become an important therapeutic target. We have recently shown that thiazolides, a novel class of anti-infectious drugs, induce apoptosis in colorectal tumor cells in a GSTP1-1-dependent manner, thereby bypassing this GSTP1-1-mediated drug resistance. In this study we investigated in detail the underlying mechanism of thiazolide-induced apoptosis induction in colorectal tumor cells. Thiazolides induce the activation of p38 and Jun kinase, which is required for thiazolide-induced cell death. Activation of these MAP kinases results in increased expression of the pro-apoptotic Bcl-2 homologs Bim and Puma, which inducibly bind and sequester Mcl-1 and Bcl-xL leading to the induction of the mitochondrial apoptosis pathway. Of interest, while an increase in intracellular glutathione levels resulted in increased resistance to cisplatin, it sensitized colorectal tumor cells to thiazolide-induced apoptosis by promoting increased Jun kinase activation and Bim induction. Thus, thiazolides may represent an interesting novel class of anti-tumor agents by specifically targeting tumor resistance mechanisms, such as GSTP1-1.

N-acetylcysteine blocks apoptosis induced by N-alpha-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells

The serine protease inhibitor N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, N-acetylcysteine (NAC), as well as L- or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-kappaB activation in T. parva-transformed T-cells, with phosphorylation of IkappaBalpha and IkappaBbeta being inhibited with different kinetics. TPCK-mediated inhibition of IkappaB phosphorylation, NF-kappaB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-kappaB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-kappaB activation pathway(s).

p16-induced apoptosis in Ewing's sarcoma

The tumor suppressor p16 is a negative regulator of the cell cycle, and acts by preventing the phosphorylation of RB, which in turn prevents the progression from G1 to S phase of the cell cycle. In addition to its role in the cell cycle, p16 may also be able to induce apoptosis in some tumors. Ewing's sarcoma, a pediatric cancer of the bone and soft tissue, was used to study the ability of p16 to induce apoptosis due to the fact that p16 is often deleted in Ewing's sarcoma tumors and may play a role in the oncogenesis or progression of this disease. The purpose of these studies was to determine whether introduction of p16 into Ewing's sarcoma cells would induce apoptosis. We infected the Ewing's sarcoma cell line TC71, which does not express p16, with adenovirus- p16 (Ad-p16). Ad-p16 infection led to the production of functional p16 as measured by the induction of G1 arrest. Ad-p16 infection induced as much as a 100% increase in G1 arrest compared to untreated cells. As measured by propidium iodide (PI) and Annexin V staining, Ad-p16 was able to induce apoptosis to levels 20–30 fold higher than controls. Furthermore, Ad-p16 infection led to loss of RB protein before apoptosis could be detected. The loss of RB protein was due to post-translational degradation of RB, which was inhibited by the addition of the proteasome inhibitors PS-341 and NPI-0052. Downregulation of RB with si-RNA sensitized cells to Ad-p16-induced apoptosis, indicating that RB protects from apoptosis in this model. This study shows that p16 leads to the degradation of RB by the ubiquitin/proteasome pathway, and that this degradation may be important for the induction of apoptosis. Given that RB may protect from apoptosis in some tumors, apoptosis-inducing therapies may be enhanced in tumors which have lost RB expression, or in which RB is artificially inactivated. ^

Modulation of BCL-X$\sb{\rm L}$ is a critical determinant of c-myc induced apoptosis

The fine balance between proliferation and apoptosis plays a primary role in carcinogenesis. Proto-oncogenes that induce both proliferation and apoptosis provide a powerful inbuilt system to inhibit clonal expansion of cells with high proliferation rates. This provides a restraint to the development of neoplasms. C-myc expressing cells undergo apoptosis in low serum by an unknown mechanism. Several lines of evidence suggested that c-myc induces apoptosis by a transcriptional mechanism. However, the target genes of this program have not been fully defined. Protein synthesis inhibitors induce apoptosis in c-myc over-expressing cells at high serum levels suggesting that inhibition of synthesis of a survival factor may induce apoptosis. We show that the expression of c-myc directly correlates with an increase in the level of a survival protein, bcl-$\rm x\sb{L},$ and a decrease in the pro-apoptotic protein, bax, at both the protein and mRNA level. Furthermore, a significant decrease of the bcl-$\rm x\sb{L}$ protein levels is observed under low serum conditions. In order to investigate the mechanism of regulation of bcl-$\rm x\sb{L}$ and bax by c-myc, the bcl-x and bax promoters were cloned, sequenced and shown to contain c-myc binding sites. The chloramephenicol acetyl transferase (CAT) reporter assay was used to demonstrate activation of the bcl-x promoter by increasing levels of c-myc when co-transfected in COS cells. The bax promoter was also shown to be transrepressed in c-myc expressing cells. The role of bcl-$\rm x\sb{L}$ in apoptosis regulation in c-myc cell lines in normal and low serum was then investigated. Cells lines expressing c-myc and bcl-$\rm x\sb{L}$ were generated and were shown to be resistant to apoptosis induction in low serum. Furthermore, cell lines expressing c-myc, anti-sense bcl-$\rm x\sb{L}$ and $\beta$-galactosidase demonstrated significantly enhanced rates of apoptosis in high serum compared to c-myc Rat 1a cells. These findings suggest that c-myc activates a survival program involving bcl-$\rm x\sb{L}$ upregulation and bax downregulation. However, this survival signal is reduced under low serum conditions by the relative downregulation of bcl-$\rm x\sb{L}$ allowing for apoptosis to proceed. These data also directly demonstrates that downregulation in the level of bcl-$\rm x\sb{L}$ associated with low serum conditions is a critical determinant of c-myc induced apoptosis. ^

Distinct roles for E2F proteins in cell growth control and apoptosis

E2F transcription activity is composed of a family of heterodimers encoded by distinct genes. Through the overproduction of each of the five known E2F proteins in mammalian cells, we demonstrate that a large number of genes encoding proteins important for cell cycle regulation and DNA replication can be activated by the E2F proteins and that there are distinct specificities in the activation of these genes by individual E2F family members. Coexpression of each E2F protein with the DP1 heterodimeric partner does not significantly alter this specificity. We also find that only E2F1 overexpression induces cells to undergo apoptosis, despite the fact that at least two other E2F family members, E2F2 and E2F3, are equally capable of inducing S phase. The ability of E2F1 to induce apoptosis appears to result from the specific induction of an apoptosis-promoting activity rather than the lack of induction of a survival activity, because co-expression of E2F2 and E2F3 does not rescue cells from E2F1-mediated apoptosis. We conclude that E2F family members play distinct roles in cell cycle control and that E2F1 may function as a specific signal for the initiation of an apoptosis pathway that must normally be blocked for a productive proliferation event.

Fas-induced apoptosis of T cells occurs independently of ceramide generation

The Fas receptor is one of a number of important physiological inducers of programmed cell death (apoptosis). Current models for regulation of this process involve rapid conversion of sphingomyelin to ceramide by cellular sphingomyelinases. Induced changes in cellular levels of such sphingosine-based ceramides are normally extrapolated from measurements of sphingomyelinase activity or following their conversion to ceramide phosphate by treatment of cellular lipid extracts with bacterial diacylglycerol kinase (DAGK). To allow direct study of cellular sphingosine- and sphinganine-based ceramide levels, we developed a mass spectrometric technique capable of determining inducible changes in both overall ceramide levels and species distribution in cellular lipid preparations. Contrary to current models, we detected no changes in cellular ceramide levels up to 2 hr poststimulation of Jurkat T cells with an anti-Fas IgM, although this treatment did induce apoptosis. We also determined in the same system that, when utilizing the DAGK assay, increased phosphorylation of substrates that comigrated with ceramide standards was apparent but that this effect was due to an enhancement of DAGK activity rather than increases in levels of cellular ceramides as substrates per se. Thus, the first direct measurement of ceramides present in cells undergoing apoptosis indicates that, insofar as it can be measured, the induction of apoptosis does not involve the generation of sphingosine-based ceramides, contrary to many published accounts.

c-Abl-induced apoptosis, but not cell cycle arrest, requires mitogen-activated protein kinase kinase 6 activation

c-Abl is a ubiquitously expressed protein tyrosine kinase activated by DNA damage and implicated in two responses: cell cycle arrest and apoptosis. The downstream pathways by which c-Abl induces these responses remain unclear. We examined the effect of overexpression of c-Abl on the activation of mitogen-activated protein kinase pathways and found that overexpression of c-Abl selectively stimulated p38, while having no effect on c-Jun N-terminal kinase or on extracellular signal-regulated kinase. c-Abl-induced p38 activation was primarily mediated by mitogen-activated protein kinase kinase (MKK)6. A C-terminal truncation mutant of c-Abl showed no activity for stimulating p38 and MKK6, while a kinase-deficient c-Abl mutant still retained a residual activity. We tested different forms of c-Abl for their ability to induce apoptosis and found that apoptosis induction correlated with the activation of the MKK6-p38 kinase pathway. Importantly, dominant-negative MKK6, but not dominant-negative MKK3 or p38, blocked c-Abl-induced apoptosis. Because overexpression of p38 blocks cell cycle G1/S transition, we also tested whether the MKK6-p38 pathway is required for c-Abl-induced cell cycle arrest, and we found that neither MKK6 nor p38 dominant-negative mutants could relieve c-Abl-induced cell cycle arrest. Finally, DNA damage-induced MKK6 and p38 activation was diminished in c-Abl null fibroblasts. Our study suggests that c-Abl is required for DNA damage-induced MKK6 and p38 activation, and that activation of MKK6 by c-Abl is required for c-Abl-induced apoptosis but not c-Abl-induced cell cycle arrest.

The intrinsic radioresistance of glioblastoma-derived cell lines is associated with a failure of p53 to induce p21BAX expression

Radiation is the primary modality of therapy for all commonly occurring malignant brain tumors, including medulloblastoma and glioblastoma. These two brain tumors, however, have a distinctly different response to radiation therapy. Medulloblastoma is very sensitive to radiation therapy, whereas glioblastoma is highly resistant, and the long-term survival of medulloblastoma patients exceeds 50%, while there are few long-term survivors among glioblastoma patients. p53-mediated apoptosis is thought to be an important mechanism mediating the cytotoxic response of tumors to radiotherapy. In this study, we compared the response to radiation of five cell lines that have wild-type p53: three derived from glioblastoma and two derived from medulloblastoma. We found that the medulloblastoma-derived cell lines underwent extensive radiation-induced apoptotic cell death, while those from glioblastomas did not exhibit significant radiation-induced apoptosis. p53-mediated induction of p21BAX is thought to be a key component of the pathway mediating apoptosis after the exposure of cells to cytotoxins, and the expression of mRNA encoding p21BAX was correlated with these cell lines undergoing radiation-induced apoptosis. The failure of p53 to induce p21BAX expression in glioblastoma-derived cell lines is likely to be of biologic significance, since inhibition of p21BAX induction in medulloblastoma resulted in a loss of radiation-induced apoptosis, while forced expression of p21BAX in glioblastoma was sufficient to induce apoptosis. The failure of p53 to induce p21BAX in glioblastoma-derived cell lines suggests a distinct mechanism of radioresistance and may represent a critical factor in determining therapeutic responsiveness to radiation in glioblastomas.

Hepatitis B virus X protein and p53 tumor suppressor interactions in the modulation of apoptosis

We have reported previously that the hepatitis B virus oncoprotein, HBx, can bind to the C terminus of p53 and inhibit several critical p53-mediated cellular processes, including DNA sequence-specific binding, transcriptional transactivation, and apoptosis. Recognizing the importance of p53-mediated apoptosis for maintaining homeostasis and preventing neoplastic transformation, here we further examine the physical interaction between HBx and p53 as well as the functional consequences of this association. In vitro binding studies indicate that the ayw and adr viral subtypes of HBx bind similar amounts of glutathione S-transferase-p53 with the distal C terminus of HBx (from residues 111 to 154) being critical for this interaction. Using a microinjection technique, we show that this same C-terminal region of HBx is necessary for sequestering p53 in the cytoplasm and abrogating p53-mediated apoptosis. The transcriptional transactivation domain of HBx also maps to its C terminus; however, a comparison of the ability of full-length and truncated HBx protein to abrogate p53-induced apoptosis versus transactivate simian virus 40- or human nitric oxide synthase-2 promoter-driven reporter constructs indicates that these two functional properties are distinct and thus may contribute to hepatocarcinogenesis differently. Collectively, our data indicate that the distal C-terminal domain of HBx, independent of its transactivation activity, complexes with p53 in the cytoplasm, partially preventing its nuclear entry and ability to induce apoptosis. These pathobiological effects of HBx may contribute to the early stages of hepatocellular carcinogenesis.

The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1

Recently, Salmonella spp. were shown to induce apoptosis in infected macrophages. The mechanism responsible for this process is unknown. In this report, we establish that the Inv-Spa type III secretion apparatus target invasin SipB is necessary and sufficient for the induction of apoptosis. Purified SipB microinjected into macrophages led to cell death. Binding studies show that SipB associates with the proapoptotic protease caspase-1. This interaction results in the activation of caspase-1, as seen in its proteolytic maturation and the processing of its substrate interleukin-1β. Caspase-1 activity is essential for the cytotoxicity. Functional inhibition of caspase-1 activity by acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone blocks macrophage cytotoxicity, and macrophages lacking caspase-1 are not susceptible to Salmonella-induced apoptosis. Taken together, the data demonstrate that SipB functions as an analog of the Shigella invasin IpaB.