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 transformation-dependent 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.

Phosphoinositide 3-kinase is involved in the tumor-specific activation of human breast cancer cell Na(+)/H(+) exchange, motility, and invasion induced by serum deprivation.

Whereas the tumor acidic extracellular pH plays a crucial role in the invasive process, the mechanism(s) behind this acidification, especially in low nutrient conditions, are unclear. The regulation of the Na(+)/H(+) exchanger (NHE) and invasion by serum deprivation were studied in a series of breast epithelial cell lines representing progression from non-tumor to highly metastatic cells. Whereas serum deprivation reduced lactate production in all three cells lines, it inhibited NHE activity in the non-tumor cells and stimulated it in the tumor cells with a larger stimulation in the metastatic cells. The stimulation of NHE in the tumor cell lines was the result of an increased affinity of the internal H(+) regulatory site of the NHE without changes in sodium kinetics or expression. Serum deprivation conferred increased cell motility and invasive ability that were abrogated by specific inhibition of the NHE. Inhibition of phosphoinositide 3-kinase by overexpression of a dominant-negative mutant or wortmannin incubation inhibited NHE activity and invasion in serum replete conditions while potentiating the serum deprivation-dependent activation of the NHE and invasion. These results indicate that the up-regulation of the NHE by a phosphoinositide 3-kinase-dependent mechanism plays an essential role in increased tumor cell invasion induced by serum deprivation.

Carotenoids inhibit DNA synthesis in human aortic smooth muscle cells.

Quiescent, serum-starved human aortic smooth muscle cells were restimulated with 20% foetal calf serum in Dulbecco's modified Eagle medium, in the presence and absence of beta-carotene, canthaxanthin, zeaxanthin, lycopene, lutein or beta-cryptoxanthin, at final concentrations up to 23 microM. Concentration-dependent inhibition of DNA synthesis, measured by [methyl-3H]thymidine incorporation, was observed for the carotenoids, except for canthaxanthin and lutein which had no effect. Lycopene was the most potent of the carotenoids tested. The results suggest that antiproliferative effects of dietary carotenoids might be of significance in vivo.

The CXXC Zn binding motifs of the human papillomavirus type 16 E7 oncoprotein are not required for its in vitro transforming activity in rodent cells.

The conserved region 3 (CR3) of the E7 protein of human papillomaviruses contains two CXXC motifs involved in zinc binding and in the homodimerization of the molecule. Studies have suggested that the intact CXXC motifs in the CR3 of HPV16 and HPV18 E7 are required for the in vitro transforming activity of these proteins. CR3 also contains a low affinity pRb binding site and is involved in the disruption of the E2F/Rb1 complex. E7 is structurally and functionally related to Adenovirus E1A protein, which also has two CXXC motifs in CR3. However, the Ad E1A transforming activity appears to be independent of the presence of such domains. In fact, this viral protein exists in vivo as two different forms of 289 and 243 amino acids. The shorter Ad E1A form (Ad E1A243), where both CXXC motifs are deleted by internal splicing, retains its in vitro transforming activity. We have investigated if the HPV16 E7 CR3 can be functionally replaced by the Ad E1A243 CR3, which lacks both CXXC motifs. A chimeric protein (E7/E1A243) containing the CR1 and CR2 of HPV16 E7 fused to the CR3 of Ad E1A 243 was constructed. The E7/E1A243 while not able to homodimerize in the S. cerevisiae two-hybrid system retains several of the properties of the parental proteins, HPV16 E7 and Ad E1A. It associates with the 'pocket' proteins, induces growth in soft agar of NIH3T3 cells and immortalizes rat embryo fibroblasts. These data suggest that the CXXC motifs in CR3 of E7 do not play a direct role in the transforming properties of this viral protein but probably are important for maintaining the correct protein configuration.

Gonadotrophin releasing hormone (GnRH) receptor and steroid receptors in human uterine leiomyoma, myometrium and endometrium.

The present study evaluated the presence of GnRH-R in leiomyomas, in associated, non-involved uterine tissues (myometrium and endometrium) and the possible relationships between GnRH-R and the receptors for estrogen and progesterone in the same tissues. GnRH-R was found in all uterine tissues and both GnRH and the GnRH analog, goserelin, displaced its binding consistent with a single type of high affinity receptor (Kd approximate to 10(-8) M). GnRH-R were found more frequently in myometrium (81% of samples) than in endometrium (58%) or leiomyoma (42%). However, the mean receptor content was lowest in myometrium (139+/-19 fmol/mg protein) with both leiomyomas (288+/-77 fmol/mg protein) and endometrium (372+/-96 fmol/mg protein) having significantly higher values. Endometrial GnRH binding varied from 596+/-42 in uteri that were GnRH-R positive in the endothelium alone to 231+/-49 when GnRH-R was present also in the other tissues. Endometrium negative for the GnRH-R had significantly higher levels of estrogen receptor than all the other uterine samples (266+/-25 vs 61+/-7.5 fmol/mg protein, respectively). Endometrial GnRH-R seem to be dependent on its presence and/or level in other uterine tissues. Further, when GnRH-R is absent in the endometrium this tissue expresses greatly increased levels of steroid receptors.

Characterization of a h-3 GnRH method for the measurement of GnRH binding-sites in human breast-cancer and breast-cancer cell-lines.

The peptide hormone, GnRH has been hypothesized to play a direct antitumoral role in certain kinds of breast cancer. This direct effect is considered to function via high affinity membrane receptors to GnRH that have been demonstrated in tumors. However, no routine assay for GnRH receptors exist limiting further research in elucidating the mechanisms of GnRH action during treatment and both the clinical usefulness of this measure and the development or refinement of GnRH treatments. Part of the problem has been that previously reported procedures require expensive, difficult (for routine clinical laboratories) iodination of specific GnRH analogs. The use of iodinated substrates present the additional problems of radioactive waste disposal and safety of laboratory personal. Further, the use of different analogs complicates the comparison of data between laboratories. We present an assay that utilizes tritiated natural GnRH in order to assay the native receptor binding and reduce both safety problems and cost of assay/waste disposal. The use of nitrocellulose filters treated overnight with 1% BSA resolved the problems of high blank values. Kinetic data demonstrate that this assay is sensitive, accurate and give results comparable with other methods. We also show that the membranes derived from the tumor and the cell cultures may be frozen at -80 degrees C for up to 90 days without affecting the results of the assay.

HER-2/neu gene in primary and local metastatic axillary lymph nodes in human breast tumors.

In order to verify whether the HER-2/neu gene is involved in the initial phases of neoplastic disease or in its progression, we evaluated the amplification and overexpression of this gene in the primary tumor and in synchronous metastatic axillary lymph nodes of 26 women with operable breast cancer. HER-2/neu was amplified in 35% and overexpressed in 33% of the primary sites; similar percentages were found in lymph nodes. The clear correlation between the two disease sites regarding gene, mRNA and protein levels, supports the hypothesis that this gene is involved in the initial and invasive phases of neoplasia. Its actual role with respect to other biological tumor characteristics during the metastatic process should be investigated further.