Prolactin receptor is a negative prognostic factor in patients with squamous cell carcinoma of the head and neck.

BACKGROUND: The influence of human prolactin (hPRL) on the development of breast and other types of cancer is well established. Little information, however, exists on the effects of hPRL on squamous cell carcinomas of the head and neck (SCCHNs). METHODS: In this study, we evaluated prolactin receptor (PRLR) expression in SCCHN cell lines and assessed by immunohistochemistry the expression in 89 patients with SCCHNs. The PRLR expression was correlated with clinicopathological characteristics as well as clinical outcome. The effect of hPRL treatment on tumour cell growth was evaluated in vitro. RESULTS: Immunoreactivity for PRLR was observed in 85 out of 89 (95%) tumours. Multivariate COX regression analysis confirmed high levels of PRLR expression (>25% of tumour cells) to be an independent prognostic factor with respect to overall survival (HR=3.70, 95% CI: 1.14-12.01; P=0.029) and disease-free survival (P=0.017). Growth of PRLR-positive cancer cells increased in response to hPRL treatment. CONCLUSION: Our data indicate that hPRL is an important growth factor for SCCHN. Because of PRLR expression in a vast majority of tumour specimens and its negative impact on overall survival, the receptor represents a novel prognosticator and a promising drug target for patients with SCCHNs.

Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells.

Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.

A role for mitochondrial Bak in apoptotic response to anticancer drugs.

In the present study a clonal Jurkat cell line deficient in expression of Bak was used to analyze the role of Bak in cytochrome c release from mitochondria. The Bak-deficient T leukemic cells were resistant to apoptosis induced by UV, staurosporin, VP-16, bleomycin, or cisplatin. In contrast to wild type Jurkat cells, these Bak-deficient cells did not respond to UV or treatment with these anticancer drugs by membranous phosphatidylserine exposure, DNA breaks, activation of caspases, or release of mitochondrial cytochrome c. The block in the apoptotic cascade was in the mitochondrial mechanism for cytochrome c release because purified mitochondria from Bak-deficient cells failed to release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. The resistance of Bak-deficient cells to VP-16 was reversed by transduction of the Bak gene into these cells. Also, the cytochrome c releasing capability of the Bak-deficient mitochondria was restored by insertion of recombinant Bak protein into purified mitochondria. Following mitochondrial localization, low dose recombinant Bak restored the mitochondrial release of cytochrome c in response to Bax; at increased doses it induced cytochrome c release itself. The function of Bak is independent of Bid and Bax because recombinant Bak induced cytochrome c release from mitochondria purified from Bax(-/-), Bid(-/-), or Bid(-/-) Bax(-/-) mice. Together, our findings suggest that Bak plays a key role in the apoptotic machinery of cytochrome c release and thus in the chemoresistance of human T leukemic cells.

Comparative biochemical and immunocytochemical studies reveal differences in the effects of Clostridium perfringens enterotoxin on polarized CaCo-2 cells versus Vero cells.

Since most in vitro studies exploring the action of Clostridium perfringens enterotoxin (CPE) utilize either Vero or CaCo-2 cells, the current study directly compared the CPE responsiveness of those two cell lines. When CPE-treated in suspension, both CaCo-2 and Vero cells formed SDS-resistant, CPE-containing complexes of approximately 135, approximately 155, and approximately 200 kDa. However, confluent Transwell cultures of either cell line CPE-treated for 20 min formed only the approximately 155-kDa complex. Since those Transwell cultures also exhibited significant (86)Rb release, approximately 155-kDa complex formation is sufficient for CPE-induced cytotoxicity. Several differences in CPE responsiveness between the two cell lines were also detected. (i) CaCo-2 cells were more sensitive when CPE-treated on their basal surface, whereas Vero cells were more sensitive when CPE-treated on their apical surface; those sensitivity differences correlated with CPE binding the apical versus basolateral surfaces of these two cell lines. (ii) CPE-treated Vero cells released (86)Rb into both Transwell chambers, whereas CaCo-2 cells released (86)Rb only into the CPE-containing Transwell chamber. (iii) Vero cells express the tight junction (TJ) protein occludin but (unlike CaCo-2 cells) cannot form TJs. The ability of TJs to affect CPE responsiveness is supported by the similar effects of CPE on Transwell cultures of CaCo-2 cells and Madin-Darby canine kidney cells, another polarized cell forming TJs. Confluent CaCo-2 Transwell cultures CPE-treated for >1 h formed the approximately 200-kDa CPE complex (which also contains occludin), exhibited morphologic damage, and had occludin removed from their TJs. Collectively, these results identify CPE as a bifunctional toxin that, in confluent polarized cells, first exerts a cytotoxic effect mediated by the approximately 155-kDa complex. Resultant damage then provides CPE access to TJs, leading to approximately 200-kDa complex formation, internalization of some TJ proteins, and TJ damage that may increase paracellular permeability and thereby contribute to the diarrhea of CPE-induced gastrointestinal disease.

Apoptosis-resistant mitochondria in T cells selected for resistance to Fas signaling.

Jurkat leukemic T cells are highly sensitive to the extrinsic pathways of apoptosis induced via the death receptor Fas or tumor necrosis factor-related apoptosis-inducing ligand as well as to the intrinsic/mitochondrial pathways of death induced by VP-16 or staurosporin. We report here that clonal Jurkat cell lines selected for resistance to Fas-induced apoptosis were cross-resistant to VP-16 or staurosporin. Each of the apoptotic pathways was blocked at an apical phase, where common regulators of apoptosis have not yet been defined. The Fas pathway was blocked at the level of caspase-8, whereas the intrinsic pathway was blocked at the mitochondria. No processing or activity of caspases was detected in resistant cells in response to either Fas-cross-linking or VP-16 treatment. Also, no apoptosis-associated alterations in the mitochondrial inner membrane, outer membrane, or matrix were detected in resistant Jurkat cells treated with VP-16. Thus, no changes in permeability transition, loss in inner membrane cardiolipin, generation of reactive oxygen species, or release of cytochrome c were observed in resistant cells treated with VP-16. Further, unlike purified mitochondria from wild type cells, those obtained from resistant cells did not release cytochrome c or apoptosis-inducing factor in response to recombinant Bax or truncated Bid. These results identify a defect in mitochondria ability to release intermembrane proteins in response to Bid or Bax as a mechanism of resistance to chemotherapeuetic drugs. Further, the selection of VP-16-resistant mitochondria via elimination of Fas-susceptible cells may suggest the existence of a shared regulatory component between the extrinsic and intrinsic pathways of apoptosis.

Inhibitor of apoptosis protein hILP undergoes caspase-mediated cleavage during T lymphocyte apoptosis.

Several endogenous or viral inhibitors of apoptosis, including Bcl-2, Bcl-xL, FLIP, p35, and CrmA, have been shown to be cleaved by caspases during apoptosis. In this study, we demonstrate that the endogenous inhibitor of apoptosis, hILP/XIAP, is also cleaved in apoptotic T lymphocytes, generating at least one prominent fragment of 29 kDa. This p29 cleaved fragment was detected in Jurkat cells induced to apoptose by anti-Fas antibody, staurosporin, or VP-16. The cleavage of hILP appears to be caspase mediated because the production of the p29 protein was inhibited by the pan-caspase peptide inhibitor, Z-VAD.FMK. In Jurkat cells engineered to overexpress CrmA, cleavage of hILP in response to anti-Fas antibody or staurosporin was inhibited, whereas overexpression of Bcl-2 abrogated the cleavage in response to VP-16. Cleavage of hILP was also observed in cell-free reactions using in vitro translated hILP and recombinant caspase-3 or -7. Moreover, we found that the p29 hILP fragment retained the ability to bind caspase-3 and -7, as shown previously for full-length or BIR-2 hILP. The p29 cleavage product was also detected during T-cell receptor-mediated apoptosis in peripheral blood lymphocytes from normal donors. Furthermore, tumor-associated T lymphocytes purified from ascites of patients with ovarian cancer expressed fragmented hILP, which was not detected in control T cells purified from peripheral blood of normal donors. Our results suggest that the cleavage of hILP represents an important event in apoptosis of T lymphocytes in both normal and pathological in vivo settings.

Tumor-induced apoptosis of T cells: amplification by a mitochondrial cascade.

We have recently reported that apoptosis of T cells induced by squamous cell carcinoma of the head and neck (SCCHN) is partly Fas dependent. This tumor-induced T-cell death is mediated by the activities of caspase-8 and caspase-3 and is partially inhibited by antibodies to either Fas or Fas ligand. We report here that in contrast to apoptosis induced by agonistic anti-Fas antibody (Ab), the tumor-induced apoptotic cascade in Jurkat cells is significantly amplified by a mitochondrial loop. The involvement of mitochondria in tumor-induced apoptosis of T cells was demonstrated by changes in mitochondrial permeability transition as assessed by 3,3'-dihexiloxadicarbocyanine staining, by cleavage of cytosolic BID and its translocation to the mitochondria, by release of cytochrome c to the cytosol, and by the presence of active subunits of caspase-9 in Jurkat T cells cocultured with tumor cells. To further elucidate the significance of mitochondria in tumor-induced T-cell death, we investigated the effects of various inhibitors of the mitochondrial pathway. Specific antioxidants, as well as two inhibitors of mitochondria permeability transition, bongkrekic acid and cyclosporin A, significantly blocked the DNA degradation induced in Jurkat T cells by SCCHN cells. However, these inhibitors had no effect on cells triggered by anti-Fas Ab. Furthermore, a cell-permeable inhibitor of caspase-9, Ac-LEHD.CHO, which did not inhibit T-cell apoptosis induced by anti-Fas Ab, markedly inhibited apoptosis induced by etoposide or by coculture of Jurkat with SCCHN cells. These findings demonstrate that apoptotic cascades induced in Jurkat T lymphocytes by anti-Fas Ab or tumor cells are differentially susceptible to a panel of inhibitors of mitochondrial apoptotic events. It appears that besides the Fas-mediated pathway, additional mitochondria-dependent cascades are involved in apoptosis of tumor-associated lymphocytes. Inhibition of mitochondria-dependent cascades of caspase activation should be considered to enhance the success of immunotherapy or vaccination protocols in cancer.

Identification of a Clostridium perfringens enterotoxin region required for large complex formation and cytotoxicity by random mutagenesis.

Clostridium perfringens enterotoxin (CPE), a single polypeptide of 319 amino acids, has a unique multistep mechanism of action. In the first step, CPE binds to claudin proteins and/or a 50-kDa eukaryotic membrane protein receptor, forming a small ( approximately 90-kDa) complex. This small complex apparently then associates with a 70-kDa eukaryotic membrane protein, resulting in formation of a large complex that induces the onset of membrane permeability alterations. To better define the boundaries of CPE functional regions and to identify specific amino acid residues involved in various steps of CPE action, in this study we subjected the cloned cpe gene to random mutagenesis in XL-1 Red strains of Escherichia coli. Seven CPE random mutants with reduced cytotoxicity for Vero cells were phenotypically characterized for the ability to complete each step in CPE action. Five of these seven recombinant CPE (rCPE) random mutants (G49D, S59L, R116S, R137G, and S167P) exhibited binding characteristics similar to those of rCPE or native CPE, while the Y310C and W226Stop mutants showed reduced binding and no binding, respectively, to brush border membranes. Interestingly, two completely nontoxic mutants (G49D and S59L) were able to bind and form small complex but they did not mediate any detectable large complex formation. Another strongly attenuated mutant, R116S, formed reduced amounts of an anomalously migrating large complex. Collectively, these results provide further support for large complex formation being an essential step in CPE action and also identify the CPE region ranging from residues approximately 45 to 116 as important for large complex formation. Finally, we also report that limited removal of extreme N-terminal CPE sequences, which may occur in vivo during disease, stimulates cytotoxic activity by enhancing large complex formation.

Characterization of membrane-associated Clostridium perfringens enterotoxin following pronase treatment.

After binding, Clostridium perfringens enterotoxin (CPE) initially localizes in a small (approximately 90-kDa) complex in plasma membranes. This event is followed by formation of a second membrane complex, referred to as large (160-kDa) complex. Contrary to a previous hypothesis proposing that CPE inserts into intestinal brush border membranes (BBMs) when this toxin is localized in the small complex, this study shows that BBMs do not offer CPE localized in the small complex protection from pronase. However, our experiments indicate that BBMs do substantially protect CPE from pronase when this toxin is localized in large complex. Since the onset of CPE-induced permeability alterations closely coincides with large-complex formation, these new results suggest that CPE-induced alterations in permeability may result from pore formation due to the partial membrane insertion of CPE when this toxin is present in large complex.

Clostridium perfringens type E animal enteritis isolates with highly conserved, silent enterotoxin gene sequences.

Several Clostridium perfringens genotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express alpha and iota toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringens enterotoxin (CPE), were unable to express CPE. These silent cpe sequences were shown to be highly conserved among type E isolates. However, relative to the functional cpe gene of type A isolates, these silent type E cpe sequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpe sequences do not appear to be clonally related, and their silent type E cpe sequences are always located, near the iota toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpe sequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries iota toxin genes, to several different type A C. perfringens isolates.

Evidence that an approximately 50-kDa mammalian plasma membrane protein with receptor-like properties mediates the amphiphilicity of specifically bound Clostridium perfringens enterotoxin.

Previous studies suggest that Clostridium perfringens enterotoxin (CPE) inserts into mammalian membranes. Using Triton X-114 phase separation analysis and charge-shift electrophoresis, this study demonstrates that CPE exhibits the amphiphilicity required for membrane insertion, but this behavior develops only after exposure of CPE to membranes. This effect does not require proteolytic or covalent CPE modifications or formation of a previously reported 160-kDa CPE-containing complex. A novel 90-kDa CPE-containing complex with amphiphilic properties was detected in intestinal brush-border membranes and in CPE-sensitive, but not CPE-insensitive, cell lines using nondenaturing Triton X-100 electrophoresis. Immunoprecipitation analysis suggested that the 90-kDa complex is composed of CPE and a 45-50-kDa membrane protein. Since the 90-kDa complex is formed only in cells that bind and respond to CPE, these results are consistent with the 45-50-kDa protein mediating CPE amphiphilicity and serving as a functional CPE receptor. A four-step model for CPE action is proposed. 1) CPE binds to the 45-50-kDa protein to form a 90-kDa complex. 2) The 90-kDa complex undergoes some physical change corresponding to insertion or a conformational change. 3) The 90-kDa complex and a 70-kDa membrane protein interact to form a 160-kDa complex. 4) Formation of the 160-kDa complex leads to permeability alterations.

Mapping of functional regions of Clostridium perfringens type A enterotoxin.

Studies were conducted to allow construction of an initial map of the structure-versus-function relationship of the Clostridium perfringens type A enterotoxin (CPE). Removal of the N-terminal 25 amino acids of CPE increased the primary cytotoxic effect of CPE but did not affect binding. CPE sequences required for at least four epitopes were also identified.