Massimo Tommasino

Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associated phenotypes

In this study we investigate the mechanism of intracellular pH change and its role in malignant transformation using the E7 oncogene of human papillomavirus type 16 (HPV16). Infecting NIH3T3 cells with recombinant retroviruses expressing the HPV16 E7 or a transformation deficient mutant we show that alkalinization is transformation specific. In NIH3T3 cells in which transformation can be turned on and followed by induction of the HPV16 E7 oncogene expression, we demonstrate that cytoplasmic alkalinization is an early event and was driven by stimulation of Na+/H+ exchanger activity via an increase in the affinity of the intracellular NHE‐1 proton regulatory site. Annulment of the E7‐induced cytoplasmic alkalinization by specific inhibition of the NHE‐1, acidification of culture medium, or clamping the pHi to nontransformed levels prevented the development of later transformed phenotypes such as increased growth rate, serum‐independent growth, anchorage‐independent growth, and glycolytic metabolism. These findings were verified in human keratinocytes (HPKIA), the natural host of HPV. Results from both NIH3T3 and HPKIA cells show that alkalinization acts on pathways that are independent of the E2F‐mediated transcriptional activation of cell cycle regulator genes. Moreover, we show that the transformationdependent increase in proliferation is independent of the concomitant stimulation of glycolysis. Finally, treatment of nude mice with the specific inhibitor of NHE‐1, DMA, delayed the development of HPV16‐keratinocyte tumors. Our data confirm that activation of the NHE‐1 and resulting cellular alkalinization is a key mechanism in oncogenic transformation and is necessary for the development and maintenance of the transformed phenotype.—Reshkin, S. J., Bellizzi, A., Caldeira, S., Albarani, V., Malanchi, I., Poignee, M., Alunni‐Fabbroni, M., Casavola, V., Tommasino, M. Na+/H+ exchanger‐dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation‐associated phenotypes.

Induction of pRb Degradation by the Human Papillomavirus Type 16 E7 Protein Is Essential To Efficiently Overcome p16INK4a-Imposed G1 Cell Cycle Arrest

ABSTRACT It has previously been shown that the E7 protein from the cutaneous human papillomavirus type 1 (HPV1), which is associated with benign skin lesions, binds the product of the tumor suppressor gene retinoblastoma (pRb) with an efficiency similar to that of the E7 protein from the oncogenic HPV type 16. Despite this ability, HPV1 E7 does not display any activity in transforming primary cells. In addition, the two viral proteins differ in their mechanisms of targeting pRb. HPV16 E7 promotes pRb destabilization, while cells expressing HPV1 E7 do not show any decrease in pRb levels. In this study, we show that HPV1 E7, in contrast to HPV16 E7, has only a weak activity to neutralize the effect of cyclin-dependent kinase inhibitor p16INK4a. By generation of HPV1/16 E7 chimeric proteins, we have identified a central motif in the two E7 proteins, which determines their different abilities to overcome the p16INK4a-mediated cell cycle arrest. This motif is located downstream of the pRb-binding domain and comprises only three amino acids in HPV16 E7. Swapping this central motif in the two viral proteins causes an exchange of their activities involved in circumventing the inhibitory function of p16INK4a. Most importantly, our data show that the efficiency of the E7 proteins in neutralizing the inhibitory effect of p16INK4a correlates with their ability to promote pRb degradation.

Increased risk for cervical disease progression of French women infected with the human papillomavirus type 16 E6-350G variant.

To test the significance of human papillomavirus (HPV) type 16 and HPV16 E6 variants as risk factors for viral persistence and progression to high-grade lesion, we did a nested case-control study within a cohort study of >15,000 Caucasian French women. Three groups infected with high-risk HPV were compared: (a) women with cleared infection (controls, n = 201), (b) women with persistent infection (cases, n = 87), and (c) women who progressed into high-grade lesion (cases, n = 58). Women with persistent HPV infection and those that progressed into high-grade lesions were likelier to harbor HPV16 than other high-risk HPV types [odds ratio (OR), 2.4; 95% confidence interval (95% CI), 1.3-4.3 and OR, 4.2; 95% CI, 2.2-8.1, respectively]. Notably, especially elevated ORs of persistence (3.0; 95% CI, 1.4-6.7) and progression (6.2; 95% CI, 2.7-14.3) were found among women who harbored the HPV16 350G variant. Thus, HPV type and HPV16 variant seem to be risk factors for viral persistence and progression of infections into high-grade cervical lesions. Cancer Epidemiol Biomarkers Prev 2006:15(4);820–2

The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin

The protein which can be labelled by low concentrations of dicyclohexylcarbodiimide in the Mr region of 30 000-35 000 has been purified from pig heart mitochondria with a high yield and as a single band of apparent Mr 35 000 in dodecyl sulphate-containing gels. The protein is not identical with the phosphate carrier as suggested before, since the two proteins behave differently during isolation. Incorporation of the isolated 35 kDa dicyclohexylcarbodiimide-binding protein into lipid bilayer membranes causes an increase of the membrane conductance in definite steps, due to the formation of pores. The specific pore-forming activity increases during the purification procedure. The single pore conductance is about 4.0 nS, suggesting a diameter of 1.7 nm of the open pore. The pore conductance is dependent on the voltage across the membrane. Anion permeability of the pore is higher than cation permeability. These properties are similar to those described for isolated mitochondrial and bacterial porins. It is concluded that the 35 kDa dicyclohexylcarbodiimide-binding protein from pig heart mitochondria is identical with porin from outer mitochondrial membrane.

Development of a sensitive and specific multiplex PCR method combined with DNA microarray primer extension to detect beta-papillomavirus types

ABSTRACT Emerging lines of evidence indicate that the cutaneous human papillomavirus (HPV) types that belong to the genus Betapapillomavirus (beta HPV) are involved in the development of nonmelanoma skin cancer. Unlike the situation for mucosal HPV types, highly sensitive and reliable methods to identify characterized cutaneous HPV types in a single assay are limited. Here, we describe a novel one-shot method for the detection of all characterized beta HPV types, namely, HPV type 5 (HPV5), 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96. This assay combines two different techniques: multiplex PCR using HPV type-specific primers for amplification of each E7 gene and array primer extension (APEX) for typing. This method has been validated using clinical samples which were analyzed simultaneously for the presence of cutaneous HPV types by two additional methods, i.e., the FAP59/64 PCR protocol and a commercially available PCR-reverse hybridization assay (PM-PCR RHA). Our data show good agreement between the results obtained with the multiplex PCR/APEX assay and the PM-PCR RHA method (overall HPV positivity of 92.2% for multiplex PCR/APEX assay versus 90.6% with the PM-PCR RHA) (kappa value, 50; 95% confidence interval, 13 to 88). In addition, the multiplex PCR/APEX assay showed higher sensitivity than the PM-PCR RHA did. This favorable feature and the high-throughput potential make this assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of cutaneous types in skin cancer.

Induction of S phase and apoptosis by the human papillomavirus type 16 E7 protein are separable events in immortalized rodent fibroblasts.

The HPV16 E7 oncoprotein neutralizes several cell cycle checkpoints, favouring the entry of quiescent cells into S phase. This activity is mediated in part by association of E7 with the pocket proteins and consequent activation of E2F transcription factors. In addition, HPV16 E7 protein is able to promote apoptosis. In this study we demonstrate that the ability to induce apoptosis is a common property of E7s belonging to both benign and malignant HPV types. The E7-induced apoptosis is mediated by inactivation of pRb, whilst neutralization of the other two pRB-related proteins, p107 and 130, is not sufficient to trigger apoptosis. Moreover, we show that certain point mutations in the conserved regionu20091 (CR1) of HPV16 E7 abolish the induction of apoptosis without altering the ability to stimulate S phase. Thus, these two E7-mediated cellular events, apoptosis and S phase entry, can be separated in immortalized rodent fibroblasts. Our findings demonstrate that the E7-mediated pRb destabilization is not required for its ability to drive quiescent cells into S phase and to induce apoptosis. Finally, expression of E7 proteins in NIH3T3, which lack a functional p19ARF, does not lead to p53 accumulation, indicating that the E7 impacts upon additional cellular pathways to promote apoptosis.

Identification of a novel activity of human papillomavirus type 16 E6 protein in deregulating the G1/S transition.

In this study we show that E6 of human papillomavirus has the ability to deregulate the cell cycle G1/S transition. In rodent immortalized fibroblasts (NIH3T3) serum deprivation or over-expression of the cyclin-dependent kinase inhibitors, p16INK4a or p27KIP1, leads to G1 cell cycle arrest. HPV16 E6 overcomes the antiproliferative signals, gaining the ability to drive serum-deprived and p16INK4a or p27KIP1 over-expressing cells into S phase. E6 protein from the benign HPV type 1 displays a similar activity to HPV16 E6 to deregulate the G1/S transition. Thus, this activity appears to be conserved between E6 proteins from non-oncogenic and oncogenic HPV types. Furthermore, we show that HPV16 E6 is not able to circumvent a G1 arrest imposed by pRb mutant in which all CDK phosphorylation sites have been mutated. These data indicate that the viral protein acts upstream of pRb and its mechanism in promoting cell cycle progression is dependent on pRb phosphorylation. In summary, this study describes a novel biological function of HPV E6 and shows that the S phase entry, required for viral DNA replication, is not exclusively controlled by E7, but that E6 also is involved in this event.

Determination of the binding affinity of different human papillomavirus E7 proteins for the tumour suppressor pRb by a plate-binding assay

A plate-binding assay was developed to quantify the affinity of the E7 oncoprotein from different human papillomavirus (HPV) types for the tumour suppressor pRb. The method is highly reproducible, sensitive and easy to handle. It could be easily adapted for the quantitative study of other interacting proteins and for screenings of inhibitors of protein/protein interactions. The pRb-binding affinity of six different E7 proteins has been quantified. The K(D) values vary from approximately 4.5x10(-9) M for HPV16 E7 to more than 1x10(-7) M for HPV10 and HPV48 E7. Point mutation C24G in the high affinity pRb-binding domain of HPV16 E7 results in a 3-fold affinity reduction. The data indicate that the high affinity pRb-binding domain of E7, LXCXE, is essential for the association between the viral and cellular proteins. However, other E7 domain(s), which appear(s) not to be present in all E7s, contribute to stabilize the E7-pRb association.

A Nuclear Export Signal and Phosphorylation Regulate Dok1 Subcellular Localization and Functions

ABSTRACT Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKβ. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.

Purification of the active mitochondrial phosphate carrier by affinity chromatography with an organomercurial agarose column

The general procedure for the isolation of the phosphate carrier from mitochondria involves solubilization with non-ionic detergents and chromatography on hydroxylapatite [l-3]. With high resolution SDS-gel electrophoresis this preparation can be separated into 4-5 protein bands [2]. Further purification, in particular removal of the ADP/ATP-carrier, was obtained by chromatography on Celite [2], on Mersalyl-Ultrogel [3,4] and by using Triton X-l 14 instead of Triton X-100 [5]. However, after all these procedures the purified phosphate carrier fraction still contained 4-5 protein bands in the &.-region of 30 000-35 000 [5]. Affi-Gel 501 (an organomercurial agarose gel), Dowex AG l-X8 and hydroxylapatite (Bio-Gel HTP) were obtained from Bio-Rad; [32P]phosphate (carrier free) from Amersham; [3H]NEM from New England Nuclear; acrylamide, N,N’methylenebisacrylamide, SDS, Triton X-100 and NEM from Serva; scintillation liquid (Maxifluor) from J. Baker; L-cY-phosphatidylcholine (from egg yolk, Type X-E) from Sigma and cardiolipin from Serdary.