Physical interaction between epidermal growth factor receptor and DNA-dependent protein kinase in mammalian cells

Binding of extracellular ligands to epidermal growth factor receptors (EGFR) activate signal transduction pathways associated with cell proliferation, and these events are inhibited by monoclonal antibodies against EGFR. Since efficient DNA repair in actively growing cells may require growth factor signaling, it was of interest to explore any linkage between EGFR-mediated signaling and DNA-dependent protein kinase (DNA-PK), an enzyme believed to be involved in repairing double strand breaks and V(D)J recombination. We report that anti-EGFR monoclonal antibodies (mAbs), and not EGFR ligands, trigger a specific early physical interaction between EGFR and a 350-kDa catalytic subunit of DNA or its regulatory heterodimeric complex Ku70/80, in a variety of cell types, both in vivo and in vitro. Inhibition of EGFR signaling by anti-EGFR mAb was accompanied by a reduction in the levels of the DNA-PK and its activity in the nuclear fraction. Confocal imaging revealed that a substantial amount of DNA-PK was co-localized with EGFR in anti-EGFR mAb-treated cells. Anti-EGFR mAb-induced physical interaction between EGFR and DNA-PK or Ku70/80 was dependent on the presence of EGFR, but not on the levels of EGFR. The EGFR associated with DNA-PK or Ku70/80 retains its intrinsic kinase activity. Our findings demonstrate the existence of a novel cellular pathway in mammalian cells that involves physical interactions between EGFR and DNA-PK or Ku70/80 in response to inhibition of EGFR signaling. Our present observations suggest a possible role of EGFR signaling in maintenance of the nuclear levels of DNA-PK, and interference in EGFR signaling may possibly result in the impairment of DNA repair activity in the nuclei in anti-EGFR mAb-treated cells

Interferon-induces expression of cyclin-dependent kinase-inhibitors p21^WAF1 and p27^Kip1 that prevent activation of cyclin-dependent kinase by CDK-activating Kinase (CAK)

To understand the mechanism of Interferon (IFN)-mediated suppression of cell cycle progression, we have earlier shown that IFN-α enhances the expression of underphosphorylated retinoblastoma protein by inhibiting the cyclin-dependent kinase-2 (CDK-2) activity (Kumar and Atlas, Proc. Natl. Acad. Sci. 89, 6599 – 6603, 1992; Zhang and Kumar, Biochem. Biophysi. Res. Comm., 200, 522 – 528, 1994). In the studies presented here, we investigated the mechanism of inhibition of CDKs in IFN-treated cells by delineating the potential role(s) of CDK-inhibitors (CKIs) and CDK-activating Kinase (CAK). We report that IFN-α inhibits the H-1 kinase activity associated with CDK-4 or CDK-2 due to induction of expression of CDK-inhibitor p21^WAF1 (but not p27^Kip1) as its immunodepletion from IFN-treated extracts restored the CDK-associated H-1 kinase activity. In addition, we also show that IFN-γ induces expression of CDK-inhibitors p21^WAF1 and p27^Kip1) and inhibited the H-1 kinase activity associated with CDK-2 or CDK-4. The observed IFN-γ-mediated inhibition of CDK-2 and CDK-4 kinase activity was due to enhanced interactions with p21^WAF1 and p27^Kip1), respectively. We also demonstrated that IFN-induced CKIs prevent CAK from activating the CDK-2 as immunodepletion of induced CKIs from the inhibitory extracts resulted in the restoration of CAK-mediated activation of CDK-2

Reliability and validity of an audio signal modified shuttle walk test

Background: The audio signal in the conventionally accepted protocol of shuttle walk test (SWT) is not well-understood by the patients and modification of the audio signal may improve the performance of the test. Objectives: The aim of this study is to study the validity and reliability of an audio signal modified SWT, called the Singla-Richa modified SWT (SWTSR), in healthy normal adults. Patients and Methods: In SWTSR, the audio signal was modified with the addition of reverse counting to it. A total of 54 healthy normal adults underwent conventional SWT (CSWT) at one instance and two times SWTSRon the same day. The validity was assessed by comparing outcomes of the SWTSRto outcomes of CSWT using the Pearson correlation coefficient and Bland–Altman plot. Test-retest reliability of SWTSRwas assessed using the intraclass correlation coefficient (ICC). The acceptability of the modified test in comparison to the conventional test was assessed using Likert scale. Results: The distance walked (mean ± standard deviation) in the CSWT and SWTSRtest was 853.33 ± 217.33 m and 857.22 ± 219.56 m, respectively (Pearson correlation coefficient - 0.98; P < 0.001) indicating SWTSRto be a valid test. The SWTSRwas found to be a reliable test with ICC of 0.98 (95% confidence interval: 0.97–0.99). The acceptability of SWTSRwas significantly higher than CSWT. Conclusions: The SWTSRwith modified audio signal with reverse counting is a reliable as well as a valid test when compared with CSWT in healthy normal adults. It better understood by subjects compared to CSWT