Daman Saluja

Differential expression and activation of NF-κB family proteins during oral carcinogenesis: Role of high risk human papillomavirus infection

Oral cancer is one of the most common cancers in India and south‐east Asian region consisting of more than 50% of all malignant tumors. Along with many known risk factors, infection of Human Papillomavirus (HPV) has been associated with the development of oral cancer and is suggested to modulate host cell transcription. Reciprocally, cellular transcription factors, such as NF‐κB and AP‐1 are known to modulate the expression of viral and other genes involved in the development of cancer. In the absence of data on NF‐κB in relation to HPV in oral cancer, we studied the DNA binding activity and expression pattern of NF‐κB family of proteins in different stages of oral cancer and correlated with HPV infection that has been associated with better prognosis of the disease. A total of 110 fresh oral tissue biopsies were collected comprising 10 normal controls, 34 precancer and 66 oral cancer lesions prior to chemotherapy/radiotherapy. Diagnosis of HPV was done by both consensus and type‐specific PCR. Electrophoretic mobility shift assays, western blots and immunohistochemical analysis were performed to assess the binding activity and expression pattern of NF‐κB family of proteins (p50, p65, p52, c‐Rel, RelB and Bcl‐3) in oral tissue biopsies. Twenty seven percent (18/66) of the oral cancer biopsies showed the presence of HPV infection exclusively of high risk HPV type 16, which was primarily associated with the well differentiated squamous cell carcinomas (WDSCC). We observed a high constitutive activation of NF‐κB with concomitant upregulated expression of all the NF‐κB members in oral cancer tissues. Expression of NF‐κB components gradually increased as the severity of lesion increased from precancer to invasive cancer. NF‐κB p50 was found to be the major DNA binding component, which is indicative of homodimerization of p50 subunits. Interestingly, in HPV16 infected oral cancers although p50 showed high binding activity, p65 also showed a partial involvement as evidenced in supershift assay. Both by western blotting and immunohistochemistry, a differential overexpression and nuclear localization of p50, p65 and partially of Bcl‐3 were observed in HPV16 positive oral cancer patients that also showed an over‐expression of p21. We therefore, demonstrate a constitutive activation and differential expression of NF‐κB proteins, which change as a function of severity of oral lesions during development of oral cancer. The NF‐κB DNA binding is primarily due to homodimerization of p50 but infection of high risk HPV promotes participation of p65 in NF‐κB complex formation, leading to heterodimerization of p50/p65. We propose that the involvement of p65 in HPV infected oral cancer may be linked to improved differentiation and better prognosis of the disease when treated.

Sin3: Insight into its transcription regulatory functions

Sin3, a large acidic protein, shares structural similarity with the helix-loop-helix dimerization domain of proteins of the Myc family of transcription factors. Sin3/HDAC corepressor complex functions in transcriptional regulation of several genes and is therefore implicated in the regulation of key biological processes. Knockdown studies have confirmed the role of Sin3 in cellular proliferation, differentiation, apoptosis and cell cycle regulation, emphasizing Sin3 as an essential regulator of critical cellular events in normal and pathological processes. The present review covers the diverse functions of this master transcriptional regulator as well as illustrates the redundant and distinct functions of its two mammalian isoforms.

Transactivation and expression patterns of Jun and Fos/AP-1 super-family proteins in human oral cancer

Transcription factor activator protein‐1 (AP‐1) super‐family is known to modulate expression of array of genes during development of many cancers and considered as an important target for modern therapeutics. But the role of AP‐1 during development of human oral cancers is still poorly understood. Because oral cancer is one of the most common cancers in India and south‐east Asia, we studied the activation and expression pattern of AP‐1 family of proteins and mRNA in different stages of oral carcinogenesis. Gel‐shift assay, western blotting, immunohistochemistry and northern blotting have been used to assess the binding activity and expression pattern of AP‐1 family (c‐Jun, JunB, JunD, c‐Fos, FosB, Fra‐1 and Fra‐2) proteins and mRNA transcripts in a total of 100 fresh oral tissue specimens comprising precancer (n = 40), cancer (n = 50) and healthy control (n = 10). Constitutive activation of AP‐1 with concomitant upregulated expression of majority of AP‐1 family of proteins and mRNA was observed in cancer cases. Interestingly, almost all precancerous cases showed JunD homodimers, whereas c‐Fos/JunD was the most prevalent complex found in cancer tissues. The overexpression of EGFR mRNA, p50:p50/NF‐κB homodimer formation, together with overexpression of pERK and c‐Fos proteins in this study suggests an interesting cross talk between AP‐1 and NF‐κB pathways in oral cancers. Thus, this study demonstrates differential expression and activation of AP‐1 super‐family proteins in relation to severity of lesion and their crucial role in human oral carcinogenesis.

Characterization of protein transacetylase from human placenta as a signaling molecule calreticulin using polyphenolic peracetates as the acetyl group donors

We have earlier shown that a unique membrane-bound enzyme mediates the transfer of acetyl group(s) from polyphenolic peracetates (PA) to functional proteins, which was termed acetoxy drug: protein transacetylase (TAase) because it acted upon several classes of PA. Here, we report the purification of TAase from human placentral microsomes to homogeneity with molecular mass of 60 kDa, exhibiting varying degrees of specificity to several classes of PA confirming the structure-activity relationship for the microsome-bound TAase. The TAase catalyzed protein acetylation by a model acetoxy drug, 7,8-diacetoxy-4-methyl coumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with anti-acetyl lysine antibody. TAase activity was severely inhibited in calcium-aggregated microsomes as well as when Ca2+ was added to purified TAase, suggesting that TAase could be a calcium binding protein. Furthermore, the N-terminal sequence analysis of purified TAase (EPAVYFKEQFLD) using Swiss Prot Database perfectly matched with calreticulin (CRT), a major microsomal calcium binding protein of the endoplasmic reticulum (ER). The identity of TAase with CRT was substantiated by the observation that the purified TAase avidly reacted with commercially available antibody raised against the C-terminus of human CRT (13 residues peptide, DEEDATGQAKDEL). Purified TAase also showed Ca2+ binding and acted as a substrate for phosphorylation catalyzed by protein kinase C (PKC), which are hallmark characteristics of CRT. Further, purified placental CRT as well as the commercially procured pure CRT yielded significant TAase catalytic activity and were also found effective in mediating the acetylation of the target protein NADPH cytochrome P-450 reductase by DAMC as detected by Western blot using anti-acetyl lysine antibody. These observations for the first time convincingly attribute the transacetylase function to CRT. Hence, this transacetylase function of CRT is designated calreticulin transacetylase (CRTAase). We envisage that CRTAase plays an important role in protein modification by way of acetylation independent of Acetyl CoA.

PU.1 and partners: regulation of haematopoietic stem cell fate in normal and malignant haematopoiesis.

•  Introduction •  Transcription factor PU.1: expression distribution and function –  Functional domains of PU.1 protein –  Structure of PU.1 ETS domain and its binding to DNA –  PU.1 gene regulation •  PU.1‐interacting proteins –  NF‐IL6β (C/EBP‐δ) –  c‐Jun –  CBP –  GATA‐1 –  Antagonism between GATA‐1 and PU.1 –  Synergistic interaction between PU.1 and GATA‐1 –  C/EBP‐α –  c‐Myb –  AML‐1 –  AML‐1/ETO –  Pip/NF‐EM5/IRF‐4 –  PU.1‐protein interactions and HSC fate determination •  Concluding remarks

Tumor Suppressor Protein p53 Recruits Human Sin3B/HDAC1 Complex for Down-Regulation of Its Target Promoters in Response to Genotoxic Stress

Master regulator protein p53, popularly known as the “guardian of genome” is the hub for regulation of diverse cellular pathways. Depending on the cell type and severity of DNA damage, p53 protein mediates cell cycle arrest or apoptosis, besides activating DNA repair, which is apparently achieved by regulation of its target genes, as well as direct interaction with other proteins. p53 is known to repress target genes via multiple mechanisms one of which is via recruitment of chromatin remodelling Sin3/HDAC1/2 complex. Sin3 proteins (Sin3A and Sin3B) regulate gene expression at the chromatin-level by serving as an anchor onto which the core Sin3/HDAC complex is assembled. The Sin3/HDAC co-repressor complex can be recruited by a large number of DNA-binding transcription factors. Sin3A has been closely linked to p53 while Sin3B is considered to be a close associate of E2Fs. The theme of this study was to establish the role of Sin3B in p53-mediated gene repression. We demonstrate a direct protein-protein interaction between human p53 and Sin3B (hSin3B). Amino acids 1–399 of hSin3B protein are involved in its interaction with N-terminal region (amino acids 1–108) of p53. Genotoxic stress induced by Adriamycin treatment increases the levels of hSin3B that is recruited to the promoters of p53-target genes (HSPA8, MAD1 and CRYZ). More importantly recruitment of hSin3B and repression of the three p53-target promoters upon Adriamycin treatment were observed only in p53+/+ cell lines. Additionally an increased tri-methylation of the H3K9 residue at the promoters of HSPA8 and CRYZ was also observed following Adriamycin treatment. The present study highlights for the first time the essential role of Sin3B as an important associate of p53 in mediating the cellular responses to stress and in the transcriptional repression of genes encoding for heat shock proteins or proteins involved in regulation of cell cycle and apoptosis.

Novel function of calreticulin: Characterization of calreticulin as a transacetylase-mediating protein acetylator independent of acetyl CoA using polyphenolic acetates

Our earlier investigations culminated in the discovery of a unique membrane-bound enzyme in mammalian cells catalyzing the transfer of acetyl group from polyphenolic acetates (PAs) to certain functional proteins, resulting in the modulation of their activities. This enzyme was termed acetoxy drug:protein transacetylase (TAase) since it acted upon several classes of PAs. TAase was purified from rat liver microsomes to homogeneity and exhibited the molecular weight of 55 KDa. TAase-catalyzed protein acetylation by PAs was evidenced by the demonstration of immunoreactivity of the acetylated target protein such as nitric oxide synthase (NOS) with anti-acetyl lysine. The possible acetylation of human platelet NOS by PA as described above resulted in the enhancement of intracellular levels of nitric oxide (NO). PAs unlike the parent polyphenols were found to exhibit NO-related physiological effects. The N-terminal sequence was found to show 100 % homology with N-terminal sequence of mature calreticulin (CRT). The identity of TAase with CRT, an endoplasmic reticulum (ER) protein, was evidenced by the demonstration of the properties of CRT such as immunoreactivity with anti-calreticulin, binding to Ca2+ ions and being substrate for phosphorylation by protein kinase c (PKC), which are the hallmark characteristics of CRT. These observations for the first time convincingly attribute the transacetylase function to CRT, which possibly plays an important role in protein modification by way of carrying out acetylation of various enzymes through a biochemical mechanism independent of acetyl CoA.

Prevalence of Chlamydia infection among women visiting a gynaecology outpatient department: evaluation of an in-house PCR assay for detection of Chlamydia trachomatis

BackgroundScreening women for Chlamydia trachomatis infection in developing countries is highly desirable because of asymptomatic infection. The existing diagnostic methods in developing countries are not effective and their sensitivity fall below 45.0% which leads to further spread of infection. There is an urgent need for improved and cost effective diagnostic tests that will reduce the burden of sexually transmitted infections in the developing world.MethodsPrevalence of C. trachomatis infection among women visiting gynaecology department of Hindu Rao hospital in Delhi, India was determined using Roche Amplicor Multi Well Plate kit (MWP) as well as using in-house PCR assay. We used 593 endocervical swabs for clinical evaluation of the in-house developed assay against Direct Fluorescence Assay (DFA; Group I n = 274) and Roche Amplicor MWP kit (Group II, n = 319 samples) and determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of the in-house developed assay.ResultsWe detected 23.0% positive cases and there was a higher representation of women aged 18-33 in this group. An in-house PCR assay was developed and evaluated by targeting unique sequence within the gyrA gene of C. trachomatis. Specificity of the reaction was confirmed by using genomic DNA of human and other STI related microorganisms as template. Assay is highly sensitive and can detect as low as 10 fg of C. trachomatis DNA. The resolved sensitivity of in-house PCR was 94.5% compared with 88.0% of DFA assay. The high specificity (98.4%) and sensitivity (97.1%) of the in-house assay against Roche kit and availability of test results within 3 hours allowed for immediate treatment and reduced the risk of potential onward transmission.ConclusionsThe in-house PCR method is cost effective (~ 20.0% of Roche assay) and hence could be a better alternative for routine diagnosis of genital infection by C. trachomatis to facilitate improved screening and treatment management.

Post-transcriptional regulation of S-adenosylmethionine synthetase from its stored mRNA in germinated wheat embryos

About 2-3-fold stimulation of S-adenosylmethionine synthetase was witnessed in germinated wheat embryos (48 h). The enhancement of enzyme activity was significantly inhibited by cycloheximide and amino acid analogues. Simultaneous addition of corresponding amino acids alleviated the inhibitory effect of amino acid analogues. Conclusive proof for the de novo synthesis of S-adenosylmethionine synthetase was obtained by labelling this enzyme with [35SO4]2- in vivo. Thus de novo enzyme synthesis seemed necessary for the rise in activity of AdoMet synthetase in wheat embryos. Curiously, blocking of transcription with cordycepin failed to repress the de novo synthesis of AdoMet synthetase in germinated wheat embryos. We envisage the presence of stored mRNA for AdoMet synthetase in wheat embryos. Thus the regulation of this enzyme occurs at the post-transcriptional level. L-Methionine, which is one of the substrates of AdoMet synthetase, stimulated the enzyme activity (2-2.4-fold) over that observed in control germinated embryos. L-Methionine promotes increased de novo synthesis of AdoMet synthetase. Preincubation of enzyme fraction with L-Methionine failed to activate or stabilize the activity of AdoMet synthetase. Three isozymes of AdoMet synthetase were physically separated by DE-52 ion-exchange chromatography. One of the isozymes of AdoMet synthetase has been purified (1529-fold) to electrophoretic homogeneity by resorting to phenyl Sepharose and ATP Sepharose affinity chromatography. The purified enzyme catalyzed the synthesis of S-adenosylmethionine and also exhibited tripolyphosphatase activity. The reaction product of the purified enzyme was chemically and enzymatically characterized as S-adenosylmethionine. The molecular weight of the native enzyme is 174,000 and that of its subunit is 84,000 as determined on SDS-PAGE. Thus the native enzyme seems to be dimeric in nature.

Naringin ameliorates pentylenetetrazol-induced seizures and associated oxidative stress, inflammation, and cognitive impairment in rats: Possible mechanisms of neuroprotection

Oxidative stress and cognitive impairment are associated with PTZ-induced convulsions. Naringin is a bioflavonoid present in the grapefruit. It is a potent antioxidant, and we evaluated its effect on PTZ-induced convulsions. Rats were pretreated with normal saline, naringin (20, 40, and 80 mg/kg, i.p.), or diazepam (5mg/kg, i.p.) 30 min prior to the administration of PTZ. The administration of PTZ induced myoclonic jerks and generalized tonic-clonic seizures (GTSs). We observed that naringin significantly prolonged the induction of myoclonic jerks dose-dependently. Naringin (80 mg/kg, i.p.) pretreatment protected all rats, and this protective effect was annulled by the GABAA receptor antagonist, flumazenil. In addition, naringin reduced brain MDA and TNF-α levels and conserved GSH. The pretreatment also enhanced the performance of rats in the passive avoidance task. Our observations highlight the antioxidant, antiinflammatory, and anticonvulsant potential of naringin. Also, naringin modulates the GABAA receptor to produce anticonvulsant effects and to ameliorate cognitive impairment.