Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Limbic epilepsy in transgenic mice carrying a Ca2+/calmodulin-dependent kinase II alpha-subunit mutation.

Multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMK) phosphorylates proteins pivotally involved in diverse neuronal processes and thereby coordinates cellular responses to external stimuli that regulate intracellular Ca2+ [Hanson, P. I. & Schulman, H. (1992) Annu. Rev. Biochem. 61, 559-664]. Despite extensive study, the impact of this enzyme on control of the excitability of neuron populations in the mammalian nervous system in situ is unknown. To address this question, we studied transgenic mice carrying a null mutation (-/-) for the alpha subunit of CaMK. In contrast to wild-type littermates, null mutants exhibit profound hyperexcitability, evident in epileptic seizures involving limbic structures including the hippocampus. No evidence of increased excitability was detected in mice carrying null mutations of the gamma isoform of protein kinase C, underscoring the specificity of the effect of CaMK. CaMK plays a powerful and previously underappreciated role in control of neuronal excitability in the mammalian nervous system. These insights have important implications for analyses of mechanisms of epilepsy and, perhaps, learning and memory.

A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.

Brca1 inactivation induces p27Kip1-dependent cell cycle arrest and delayed development in the mouse mammary gland

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.

Deletions of CDKN2C in Multiple Myeloma: Biological and Clinical Implications

Purpose: Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material.Experimental Design: We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis.Results: By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor.Conclusions: Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.

Soybean lunasin mediates colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis

Colorectal cancer (CRC) is the third most common cancer worldwide. Various factors such as age, lifestyle and dietary patterns affect the risk of having CRC. Epidemiological studies showed a chemopreventive effect of soy consumption against CRC. However, which component(s) of soybean is associated with this reduced risk is not yet fully delineated. The objective of this research was to evaluate the anti-colon cancer potential of lunasin isolated from defatted soybean flour using in vitro and in vivo models of CRC. Lunasin was isolated from defatted soybean flour by a combination of different chromatographic and ultrafiltration techniques. The anti-colon cancer potential of lunasin was determined using different human colon cancer cell lines in vitro and a CRC liver metastasis model in vivo. Lunasin caused cytotoxicity to different human colon cancer cells with an IC50 value of 13.0, 21.6, 26.3 and 61.7 µM for KM12L4, RKO, HCT-116 and HT-29 human colon cancer cells, respectively. This cytotoxicity correlated with the expression of the α5 integrin on human colon cancer cells with a correlation coefficient of 0.78. The mechanism involved in the cytotoxic effect of lunasin was through cell cycle arrest and induction of the mitochondrial pathway of apoptosis. In KM12L4 human colon cancer cells, lunasin caused a G2/M phase arrest increasing the percentage of cells at G2/M phase from 12% (PBS-treated) to 24% (treated with 10 µM lunasin). This arrest was attributed to the capability of lunasin to increase the expression of cyclin dependent kinase inhibitors p21 and p27. At 10 µM, lunasin increased the expression of p21 and p27 in KM12L4 colon cancer cells by 2.2- and 2.3-fold, respectively. Flow cytometric analysis showed that lunasin at 10 µM increased the percentage of cells undergoing apoptosis from 13.6% to 24.7%. This is further supported by fluorescence microscopic analysis of KM12L4 cells treated with 10 µM lunasin showing chromatin condensation and DNA fragmentation. The mechanism involved is through modification of proteins involved in the mitochondrial pathway of apoptosis in KM12L4 cells as 10 µM lunasin reduced the expression of the anti-apoptotic Bcl-2 protein by 2-fold and increased the expression of the pro-apoptotic proteins Bax, cytochrome c and nuclear clusterin by 2.2-, 2.1- and 2.3- fold, respectively. This led to increased expression and activity of the executioner of apoptosis, caspase-3 by 1.8- and 2.3-fold, respectively. This pro-apoptotic property of lunasin can be attributed to its capability to internalize into the cytoplasm and nucleus of colon cancer cells 24 h and 72 h after treatment, respectively. In addition, lunasin mediated metastasis of colon cancer cells in vitro by inhibiting the focal adhesion kinase activation thereby reducing expression of extracellular regulated kinase and nuclear factor kappa B and finally inhibiting migration of colon cancer cells. In KM12L4 colon cancer cells, 10 µM lunasin resulted in the reduction of phosphorylation of focal adhesion kinase and extracellular regulated kinase by 2.5-fold, resulting in the reduced nuclear translocation of p50 and p65 NF-κB subunits by 3.8- and 1.4-fold, respectively. In an in vivo model of CRC liver metastasis, daily intraperitoneal administration of lunasin at 4 mg/kg body weight resulted in the inhibition of KM12L4 liver metastasis as shown by the reduction of the number of liver metastases from 28 (PBS-treated) to 14 (lunasin-treated, P = 0.047) and reduction in tumor burden as measured by liver weight/body weight from 0.13 (PBS-treated) to 0.10 (lunasin-treated, P = 0.039). Moreover, lunasin potentiated the anti-metastatic effect of the chemotherapeutic drug oxaliplatin given at 5 mg/kg body weight twice per week. Lunasin and oxaliplatin combination resulted in a more potent inhibition of outgrowth of KM12L4 cell metastases to the liver reducing the number of liver metastases by 6-fold and reducing the tumor burden in the liver by 3-fold when compared to PBS-treated group. This can be attributed by the capability of lunasin and oxaliplatin to reduce expression of proliferating cell nuclear antigen in liver-tumor tissue as measured by immunohistochemical staining. The results of this research for the first time demonstrated the anti-colon cancer potential of lunasin isolated from defatted soybean flour which might contribute to the chemopreventive effect of soybean in CRC as seen in different epidemiological studies. In conclusion, lunasin isolated from defatted soybean flour mediated colon carcinogenesis by inducing apoptosis and preventing outgrowth of metastasis. We suggest that the results of this research serve as a basis for further study on the chemopreventive effect of lunasin against CRC and a possible adjuvant role for lunasin in therapy of patients with metastatic CRC.

Evidence for u.v. induction of CDKN2 mutations in melanoma cell lines

The CDKN2 gene, encoding the cyclin dependent kinase inhibitor p16, is a tumour suppressor gene involved in melanoma and maps to chromosome band 9p22. Mutations or interstitial deletions of this gene have been found both in the germline of familial melanoma cases and somatically in melanoma cell lines. Previous mutation analyses of melanoma cell lines have indicated a high frequency of C:G to T:A transitions, with all of these mutations occurring at dipyrimidine sites. Including three melanoma cell lines carrying tandem CC to TT mutations, the spectrum of mutations so far reported indicates a possible role for u.v. radiation in the mutagenesis of this gene in some tumours. To further examine this hypothesis we have characterised mutations of the CDKN2 gene in 30 melanoma cell lines. Nineteen lines carried complete or partial homozygous deletions of the gene. Of the remaining cell lines, eight were shown by direct sequencing of PCR products from exon 1 and exon 2 to carry a total of nine different mutations of CDKN2. Two cell lines carried tandem CC to TT mutations and a high rate of C:G to T:A transitions was observed. This study provides further evidence for the role of u.v. light in the genesis of melanoma, with one target being the CDKN2 tumour suppressor gene.

Compilation of somatic mutations of the CDKN2 gene in human cancers : non-random distribution of base substitutions

The CDKN2 gene, encoding the cyclin-dependent kinase inhibitor p16, is a tumour suppressor gene that maps to chromosome band 9p21-p22. The most common mechanism of inactivation of this gene in human cancers is through homozygous deletion; however, in a smaller proportion of tumours and tumour cell lines intragenic mutations occur. In this study we have compiled a database of over 120 published point mutations in the CDKN2 gene from a wide variety of tumour types. A further 50 deletions, insertions, and splice mutations in CDKN2 have also been compiled. Furthermore, we have standardised the numbering of all mutations according to the full-length 156 amino acid form of p16. From this study we are able to define several hot spots, some of which occur at conserved residues within the ankyrin domains of p16. While many of the hotspots are shared by a number of cancers, the relative importance of each position varies, possibly reflecting the role of different carcinogens in the development of certain tumours. As reported previously, the mutational spectrum of CDKN2 in melanomas differs from that of internal malignancies and supports the involvement of UV in melanoma tumorigenesis. Notably, 52% of all substitutions in melanoma-derived samples occurred at just six nucleotide positions. Nonsense mutations comprise a comparatively high proportion of mutations present in the CDKN2 gene, and possible explanations for this are discussed.

CDKN2A/p16 is inactivated in most melanoma cell lines

The CDKN2A gene maps to chromosome 9p21-22 and is responsible for melanoma susceptibility in some families. Its product, p16, binds specifically to CDK4 and CDK6 in vitro and in vivo, inhibiting their kinase activity. CDKN2A is homozygously deleted or mutated in a large proportion of tumor cell lines and some primary tumors, including melanomas. The aim of this study was to investigate the involvement of CDKN2A and elucidate the mechanisms of p16 inactivation in a panel of 60 cell lines derived from sporadic melanomas. Twenty-six (43%) of the melanoma lines were homozygously deleted for CDKN2A, and an additional 15 (25%) lines carried missense, nonsense, or frameshift mutations. All but one of the latter group were shown by microsatellite analysis to be hemizygous for the region of 9p surrounding CDKN2A. p16 was detected by Western blotting in only five of the cell lines carrying mutations. Immunoprecipitation of p16 in these lines, followed by Western blotting to detect the coprecipitation of CDK4 and CDK6, revealed that p16 was functionally compromised in all cell lines but the one that carried a heterozygous CDKN2A mutation. In the remaining 19 lines that carried wild-type CDKN2A alleles, Western blot analysis and immunoprecipitation indicated that 11 cell lines expressed a wild-type protein. Northern blotting was performed on the remaining eight cell lines and revealed that one cell line carried an aberrantly sized RNA transcript, and two other cell lines failed to express RNA. The promoter was found to be methylated in five cell lines that expressed CDKN2A transcript but not p16. Presumably, the message seen by Northern blotting in these cell lines is the result of cross-hybridization of the total cDNA probe with the exon 1beta transcript. Microsatellite analysis revealed that the majority of these cell lines were hemi/homozygous for the region surrounding CDKN2A, indicating that the wild-type allele had been lost. In the 11 cell lines that expressed functional p16, microsatellite analysis revealed loss of heterozygosity at the markers immediately surrounding CDKN2A in five cases, and the previously characterized R24C mutation of CDK4 was identified in one of the remaining 6 lines. These data indicate that 55 of 60 (92%) melanoma cell lines demonstrated some aberration of CDKN2A or CDK4, thus suggesting that this pathway is a primary genetic target in melanoma development.

CDKN2A mutation in a non-FAMMM kindred with cancers at multiple sites results in a functionally abnormal protein

The CDKN2A gene encodes p16 (CDKN2A), a cell-cycle inhibitor protein which prevents inappropriate cell cycling and, hence, proliferation. Germ-line mutations in CDKN2A predispose to the familial atypical multiple-mole melanoma (FAMMM) syndrome but also have been seen in rare families in which only 1 or 2 individuals are affected by cutaneous malignant melanoma (CMM). We therefore sequenced exons 1alpha and 2 of CDKN2A using lymphocyte DNA isolated from index cases from 67 families with cancers at multiple sites, where the patterns of cancer did not resemble those attributable to known genes such as hMLH1, hMLH2, BRCA1, BRCA2, TP53 or other cancer susceptibility genes. We found one mutation, a mis-sense mutation resulting in a methionine to isoleucine change at codon 53 (M531) of exon 2. The individual tested had developed 2 CMMs but had no dysplastic nevi and lacked a family history of dysplastic nevi or CMM. Other family members had been diagnosed with oral cancer (2 persons), bladder cancer (1 person) and possibly gall-bladder cancer. While this mutation has been reported in Australian and North American melanoma kindreds, we did not observe it in 618 chromosomes from Scottish and Canadian controls. Functional studies revealed that the CDKN2A variant carrying the M531 change was unable to bind effectively to CDK4, showing that this mutation is of pathological significance. Our results have confirmed that CDKN2A mutations are not limited to FAMMM kindreds but also demonstrate that multi-site cancer families without melanoma are very unlikely to contain CDKN2A mutations.

The genetics of cutaneous melanoma

Although germline mutations in CDKN2A are present in approximately 25% of large multicase melanoma families, germline mutations are much rarer in the smaller melanoma families that make up most individuals reporting a family history of this disease. In addition, only three families worldwide have been reported with germline mutations in a gene other than CDKN2A (i.e., CDK4). Accordingly, current genomewide scans underway at the National Human Genome Research Institute hope to reveal linkage to one or more chromosomal regions, and ultimately lead to the identification of novel genes involved in melanoma predisposition. Both CDKN2A and PTEN have been identified as genes involved in sporadic melanoma development; however, mutations are more common in cell lines than uncultured tumors. A combination of cytogenetic, molecular, and functional studies suggests that additional genes involved in melanoma development are located to chromosomal regions 1p, 6q, 7p, 11q, and possibly also 9p and 10q. With the near completion of the human genome sequencing effort, combined with the advent of high throughput mutation analyses and new techniques including cDNA and tissue microarrays, the identification and characterization of additional genes involved in melanoma pathogenesis seem likely in the near future.

CDKN2A is not the principal target of deletions on the short arm of chromosome 9 in neuroendocrine (Merkel cell) carcinoma of the skin

The majority of small-cell lung cancers (SCLCs) express p16 but not pRb. Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC and MCC, we wished to determine if this was also the case in MCC. Twenty-nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and 1 on 9q. No loss of heterozygosity (LOH) was seen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p. Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined. Half of all informative cases had LOH at D9S168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168. A second region (IFNA-D9S126) showed LOH in 10 (44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti-p16 antibody was seen in all 11 tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti-p14(ARF) antibody. These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour-suppressor genes mapping both centromeric and telomeric of this locus.

Reduced expression of retinoblastoma gene product (pRB) and high expression of p53 are associated with poor prognosis in ovarian cancer

Paraffin sections (n = 168, 27 benign, 16 low malignant potential [LMP] and 125 malignant tumours) from epithelial ovarian tumours were evaluated immunohistochemically for expression of retinoblastoma gene product (pRB) and p53 protein, and the relationship among pRB, p53 and cyclin-dependent kinase inhibitor 2 (CDKN2) gene product p16INK4A (p16) was analysed, following our previous study of p16. Forty-one percent of the benign, 50% of the LMP and most (71%) of the malignant tumours showed high pRB expression. High expression of pRB (>50% pRB-positive cells) significantly correlated with non-mucinous histological subtypes. Reduced pRB expression, substage and residual disease were significant predictors for poor prognosis in stage I patients. All the benign and most of the LMP (81%) tumours were in either the p53-negative or low p53-positive category, but nearly half of the malignant tumours had high p53 expression. High p53 accumulation was found in non-mucinous, high grade and late stage tumours. For well-differentiated carcinomas, high p53 expression was a predictor of poor prognosis. However, even though high p53 expression was not associated with histological subtype, stage or the presence of residual disease, high p53 expression was not an independent predictor when all clinical parameters were combined. For all ovarian cancers, a close correlation was found between high p53 and high p16 expression. The relationship between the expression of pRB and p16 depended on tumour stage. In stage I tumours, high pRB was associated with low p16 reactivity. On the other hand, most advanced tumours showed both high pRB and high p16 reactivity. Int. J. Cancer 74:407–415, 1997. © 1997 Wiley-Liss, Inc.

Involvement of KLF4 in sulforaphane- and iberin-mediated induction of p21waf1/cip1

Sulforaphane (SF; 4-methylsulfinylbutyl isothiocyanate), a dietary compound derived from broccoli, may exhibit chemopreventive properties by inducing cell cycle arrest via induction of cyclin-dependent kinase inhibitor 1A (p21(waf1/cip1)), but the exact molecular mechanism has not been determined. Here we evaluate the role of the transcription factor Kruppel-like factor 4 (KLF4) in mediating the induction of p21(waf1/cip1) and cellular differentiation by SF and iberin (IB; 3-methylsulphinyl propyl isothiocyanate), also derived from broccoli. Exposure of Caco-2 and Caco-2/TC7 cells to SF and IB increased expression of both KLF4 and p21(waf1/cip1), whereas exposure of HT29 cells resulted only in induction of p21(waf1/cip1). In Caco-2 cells, small interfering RNA knock down of KLF4 expression attenuated induction of p21(waf1/cip1) in response to either SF or IB treatment. Contrary to expectation, prolonged exposure to SF reduced sucrase isomaltase activity, a marker of small intestinal differentiation in Caco-2 cells. Additional support for the SF-mediated induction of p21(waf1/cip1) by KLF4 was obtained from analyses of gastric tissue of Apc(Min/+) mice following acute intervention with SF but not from the analyses of other tissue of the intestinal tract. These results suggest that induction of p21(waf1/cip1) by SF or IB may be partly mediated by KLF4 in some colon cancer cells and tissues.

Targeting nuclear factor-kappa B to overcome resistance to chemotherapy

Intrinsic or acquired resistance to chemotherapeutic agents is a common phenomenon and a major challenge in the treatment of cancer patients. Chemoresistance is defined by a complex network of factors including multi-drug resistance proteins, reduced cellular uptake of the drug, enhanced DNA repair, intracellular drug inactivation, and evasion of apoptosis. Pre-clinical models have demonstrated that many chemotherapy drugs, such as platinum-based agents, antracyclines, and taxanes, promote the activation of the NF-κB pathway. NF-κB is a key transcription factor, playing a role in the development and progression of cancer and chemoresistance through the activation of a multitude of mediators including anti-apoptotic genes. Consequently, NF-κB has emerged as a promising anti-cancer target. Here, we describe the role of NF-κB in cancer and in the development of resistance, particularly cisplatin. Additionally, the potential benefits and disadvantages of targeting NF-κB signaling by pharmacological intervention will be addressed.