Lucy T.C. Peltenburg

A serine protease is involved in the initiation of DNA damage-induced apoptosis.

AbstractCaspases are considered to be the key effector proteases of apoptosis. Initiator caspases cleave and activate downstream executioner caspases, which are responsible for the degradation of numerous cellular substrates. We studied the role of caspases in apoptotic cell death of a human melanoma cell line. Surprisingly, the pancaspase inhibitor zVAD-fmk was unable to block cleavage of poly(ADP-ribose) polymerase (PARP) after treatment with etoposide, while it did prevent DEVDase activity. It is highly unlikely that caspase-2, which is a relatively zVAD-fmk-resistant caspase, is mediating etoposide-induced PARP cleavage, as a preferred inhibitor of this caspase could not prevent cleavage. In contrast, caspase activation and PARP degradation were blocked by pretreatment of the cells with the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). We therefore conclude that a serine protease regulates an alternative initiation mechanism that leads to caspase activation and PARP cleavage. More importantly, while zVAD-fmk could not rescue melanoma cells from etoposide-induced death, the combination with AEBSF resulted in substantial protection. This indicates that this novel pathway fulfills a critical role in the execution of etoposide-induced programmed cell death.

Relationship Between myc Oncogene Activation and MHC Class I Expression

Publisher Summary This chapter discusses the regulation of major histocompatibility complex (MHC) Class I expression in tumors and explores the oncogenes involved. The molecular mechanism of MHC Class I regulation and possible mechanisms for the action of the myc oncogenes in the down-modulation of MHC Class I expression are also discussed in the chapter. The implication of the suppression of MHC Class I by myc genes for the immune sensitivity of tumor cells and the possible mechanisms underlying it are also considered in the chapter. Members of the myc oncogene family are expressed in all vertebrate cells, where they play a key function in the regulation of growth and differentiation. Their expression increases at the onset of cell proliferation. In animal as well as in human tumor cells, the activation of certain oncogenes largely influences the expression of MHC Class I. Although the precise biological function of the coupling between c–myc expression and MHC Class I expression in normal cells is not known, this phenomenon may provide a mode of immune surveillance against tumor cells with activated myc genes.

Macrodissection versus microdissection of rectal carcinoma: minor influence of stroma cells to tumor cell gene expression profiles.

BackgroundThe molecular determinants of carcinogenesis, tumor progression and patient prognosis can be deduced from simultaneous comparison of thousands of genes by microarray analysis. However, the presence of stroma cells in surgically excised carcinoma tissues might obscure the tumor cell-specific gene expression profiles of these samples. To circumvent this complication, laser microdissection can be performed to separate tumor epithelium from the surrounding stroma and healthy tissue. In this report, we compared RNAs isolated from macrodissected, of which only surrounding healthy tissue had been removed, and microdissected rectal carcinoma samples by microarray analysis in order to determine the most reliable approach to detect the expression of tumor cell-derived genes by microarray analysis.ResultsAs microdissection yielded low tissue and RNA quantities, extra rounds of mRNA amplification were necessary to obtain sufficient RNA for microarray experiments. These second rounds of amplification influenced the gene expression profiles. Moreover, the presence of stroma cells in macrodissected samples had a minor contribution to the tumor cell gene expression profiles, which can be explained by the observation that more RNA is extracted from tumor epithelial cells than from stroma.ConclusionThese data demonstrate that the more convenient procedure of macrodissection can be adequately used and yields reliable data regarding the identification of tumor cell-specific gene expression profiles.

Transcriptional suppression of HLA-B expression by c-Myc is mediated through the core promoter elements

In melanoma, HLA class I expression is suppressed by overexpression of the c-myc oncogene. This suppression has severe consequences for the recognition of these tumor cells by the immune system of the organism. We show here that transcription of the HLA-B locus, which is mainly affected by c-Myc, is downmodulated at the level of initiation of transcription. The transcriptional activity of various HLA-B reporter constructs was tested in a melanoma cell line with low endogenous c-myc expression and in transfectants with high stable and transient c-myc expression. We demonstrated that the responsive region can be mapped to the core promoter region of HLA class I, ruling out any effects of c-myc overexpression on the enhancer A or enhancer B regions. The region subject to downregulation is confined to a 43 base pair fragment encompassing the CCAAT and TATA elements. By coupling this region to a heterologous viral enhancer, we showed that the downmodulation by c-Myc is independent of the presence and nature of an enhancer. These results suggest a mechanism in which c-Myc downregulates the expression of HLA class I genes by interfering with the basal level of transcription.

Expression and function of the apoptosis effector Apaf-1 in melanoma

Melanoma is characterised by its poor response to current therapeutic modalities. Soengas et al. showed that Apaf-1, a central component of the intrinsic apoptotic pathway, is often lost in metastatic melanoma, implying that Apaf-1 loss contributes to its remarkable resistance. In our attempts to corroborate their findings in a panel of melanoma lines, we have found no evidence for Apaf-1 downregulation as a major event. In the paper of Soengas et al., Apaf-1 loss correlates with the inability to execute an apoptotic program upon p53 activation, providing an explanation for the low frequency of p53 mutations in this tumour type. Recently, however, we found that DNA damage-induced cell death in melanoma cells can occur independently of caspase activity or cytochrome c release (not shown), and is dependent on an apical serine protease that can side-step the apoptosome to initiate cell death. Moreover, Apaf-1 and caspase-9 deficiency does not facilitate myc-induced lymphomagenesis in mice, arguing against a role for Apaf-1 loss in tumorigenesis. Together, these observations made us re-evaluate the high frequency and relevance of Apaf-1 inactivation in melanoma. We determined Apaf-1 protein levels in whole-cell lysates of 13 melanoma cell lines and three different melanocyte cultures and found that most lines, except for 607B, demonstrated higher Apaf-1 levels as compared to melanocyte cultures (Figure 1a and not shown). As Apaf-1 exerts its proapoptotic function in the cytoplasm, we also quantified Apaf-1 levels in cytosolic fractions of a selection of these melanoma cell lines. In line with the Apaf-1 levels in whole-cell extracts, cytosolic Apaf-1 levels were higher in most cell lines as compared to melanocytes (Figure 1b). This excludes the possibility that Apaf-1 is inactivated in these lines due to redistribution, similar to what has recently been reported for Burkitt’s lymphoma cell lines, and therefore indicates that loss of Apaf-1 expression is rarely found in our panel. Although our panel contains a relatively high incidence of p53 mutations (5/13), Apaf-1 loss is not correlated to p53 status. As a matter of fact, all melanoma lines that harbour wtp53 express relatively high levels of Apaf-1 (Figure 1a). The functionality of wt-p53 in these cell lines was confirmed by induction of the p53-inducible gene product p21 after triggering these cells with DNA damage (data not shown). Intriguingly, the only Apaf-1-low cell line in our panel, 607B, harbours mutant p53. These data therefore do not argue for a connection between loss of Apaf-1 and p53 mutation. We next set out to study the effect of Apaf-1 levels on caspase activation. As caspase-9 is activated by induced proximity and its cleavage is dispensable for its activity, it is not a good measure for enzymatic activity. We therefore measured cleavage of its substrate LEHD-afc in several etoposide-treated melanoma lines (Figure 1c, left panel). This analysis indicated that there is no correlation between caspase-9 activity and Apaf-1 expression (Figure 1a,c). For instance, cell line 607B expresses little Apaf-1 and displayed substantial LEHD-peptide cleavage, while 634 showed clear Apaf-1 expression and no LEHD-peptide cleavage. Besides caspase-9, which is directly regulated by Apaf-1, we also analysed effector caspase activation (DEVDase activity) that is likely to be more relevant for cell death. Also, these assays revealed no correlation between the levels of Apaf-1 and the caspase activity induced by etoposide treatment (Figure 1c, right panel). These results suggest that even though Apaf-1 may be downregulated in some melanoma lines, functional implications for such an event are not clear-cut. An attractive hypothesis that explains these observations is that an alternative, apoptosome-independent mechanism of initiating caspase activation plays an important role in melanoma. In conclusion, our data confirm that Apaf-1 levels vary in melanoma, but suggest that loss of expression may be less frequent. In this light, it is interesting to note that the Apaf-1 locus has recently been reassessed and shown to be located 40.3 Mbp centromerically on chromosome 12q. Several of the samples that were considered to be Apaf-1 LOH by Soengas et al. can therefore no longer be regarded as such when adhering to the rules set by Fujimoto. Although the discrepancy between Soengas’ and our data can possibly be explained by the type of melanoma or the treatment regimen given to the patients prior to isolation, we believe it is crucial to realise that loss of Apaf-1 does not necessarily lead to attenuation of caspase activation or of death. It is therefore of importance to nuance the concept that Apaf-1 downregulation is the key event that determines the resistance of melanoma to current treatment modalities.

p53 expression in human rectal tissue after radiotherapy: upregulation in normal mucosa versus functional loss in rectal carcinomas.

PURPOSE In vitro, ionizing radiation of epithelial cells leads to upregulation of wild-type p53 and subsequent induction of p21(waf1). The effect of radiotherapy (RT) on the expression of these proteins in patients is unknown. We assessed the influence of RT on the expression of p53 and p21(waf1) in normal mucosa and rectal carcinomas in vivo. METHODS Tumor and normal tissue samples were derived from rectal cancer patients randomized in a clinical trial in which the value of preoperative RT was evaluated. p53 and p21(waf1) expression was determined in 51 irradiated and 52 nonirradiated patients using immunohistochemistry. RESULTS In normal mucosa, both p53 and p21(waf1) were strongly upregulated after RT compared with the expression in unirradiated normal tissue (p <0.001). In tumor cells, no significant difference in the expression of p53 or p21(waf1) was found in the irradiated vs. nonirradiated group. In the few rectal tumors with wild-type p53, induction of p53 after RT did not necessarily lead to upregulation of p21(waf1). CONCLUSION These findings demonstrate that in normal mucosa, a functional p53-p21(waf1) pathway is present, whereas in tumor cells it is defective in almost all cases because of either p53 mutation or down- or upstream disruption in tumors with wild-type p53. Therefore, we believe that the role of p53 expression as a single prognostic marker in rectal cancer needs reconsideration.

REPRESSION OF THE MINIMAL HLA-B PROMOTER BY C-MYC AND P53 OCCURS THROUGH INDEPENDENT MECHANISMS

Major Histocompatibility Complex (MHC, HLA in humans) class I antigens play an important role in cellular immunology by presenting antigens to T cells. Downregulation of MHC class I expression is thought to be a mechanism by which tumor cells escape from T cell-mediated lysis. In primary human melanomas and melanoma cell lines, HLA-B expression is frequently downmodulated, correlating with elevated expression of the c-myc oncogene. Transfection experiments have shown that c-myc induces HLA-B downregulation through a -68 to +13 base pairs (bp) core promoter fragment, containing CCAAT and TATA-like (TCTA) boxes. Since (i) c-myc has been reported to activate the human p53 promoter and (ii) p53 is capable of repressing a large array of basal promoters, we investigated whether c-myc-induced HLA-B abrogation is mediated by p53. In this article, it is shown that the HLA-B core promoter is indeed repressed by wild-type p53, making p53 a candidate for mediating c-myc-induced HLA-B downregulation. However, transfection of c-myc into p53-null cell lines still resulted in suppression of the basal HLA-B promoter, demonstrating that c-myc and p53 repress the minimal HLA-B promoter through independent mechanisms.

C-Myc Represses Transiently Transfected HLA Class I Promoter Sequences not Locus-Specifically

Overexpression of the c-myc oncogene is frequently accompanied by downregulation of Major Histocompatibility Complex (MHC, HLA in humans) class I antigens. In human melanoma c-myc overexpression downmodulates HLA-B expression, whereas HLA-A is hardly affected. Repression of HLA-B is mediated through the core promoter, containing a CAAT-box and a non-conventional TATA-box. We show evidence that in transient transfection assays the HLA-A2 and HLA-B7 promoters are repressed by c-myc to the same extent. Therefore, other sequences of the HLA-A and HLA-B genes, possibly intron/exon sequences, should contribute to the locus B-specificity of the downregulation. Furthermore, c-myc does not seem to alter binding of protein complexes to the CAAT- or TATA-box of HLA-B7 or HLA-A2 in gel retardation assays. Comparison of promoters repressed by c-myc reveals a weak consensus sequence of the initiator (Inr) element: TCA(+1)YYYNY. The presence of a TCA sequence in the initiator region of the MHC class I promoter makes downregulation by c-myc through the Inr likely. We speculate that the Inr contributes to MHC class I promoter activity by stimulating recruitment of TFIID to the weak, non-conventional TATA-box, thereby making it susceptible to repression by c-myc through the Inr.

Activation of adenovirus 5 E1A transcription by region E1B in transformed primary rat cells.

The human adenovirus 5 E1A region can immortalize primary cultures of baby rat kidney cells, but requires the presence of the E1B region for complete oncogenic transformation. One of the effects of the E1B region in the transformation process is the activation of E1A expression. We have investigated the mechanism of this stimulation of E1A expression using nuclear run‐on assays with nuclei from Ad5 E1A‐ and Ad5 E1‐transformed cells. It was found that E1B enhances E1A at the level of transcription‐initiation. This activation is mainly observed when the E1A and E1B regions are integrated simultaneously into the cellular genome and only minimally when these genes are integrated separately, strongly suggesting that a close physical linkage of these regions is essential for the observed effect.

Gene expression profiling reveals two separate mechanisms regulating apoptosis in rectal carcinomas in vivo

The level of apoptosis in rectal carcinomas of patients treated by surgery only predicts local failure; patients with intrinsically high-apoptotic tumors develop less local recurrences than patients with low levels of apoptosis. To identify genes involved in this intrinsic apoptotic process in vivo, 47 rectal tumors with known apoptotic phenotype (24 low- and 23 high-apoptotic) were analyzed by oligonucleotide microarray technology. We identified several genes differentially expressed between low- and high-apoptotic tumors. Unsupervised clustering of the tumors based on expression levels of these genes separated the low-apoptotic from the high-apoptotic tumors, indicating a gene expression-dependent regulation. In addition, this clustering revealed two subgroups of high-apoptotic tumors. One high-apoptotic subgroup showed subtle differences in mRNA and protein expression of the known apoptotic regulators BAX, cIAP2 and ARC compared to the low-apoptotic tumors. The other subgroup of high-apoptotic tumors showed high expression of immune-related genes; predominantly HLA class II and chemokines, but also HLA class I and interferon-inducible genes were highly expressed. Immunohistochemistry revealed HLA-DR expression in epithelial tumor cells in 70% of these high-apoptotic tumors. The expression data suggest that high levels of apoptosis in rectal carcinoma patients can be the result of either slightly altered expression of known pro- and anti-apoptotic genes or high expression of immune-related genes.