Switching off HER-2/neu in a tetracycline-controlled mouse tumor model leads to apoptosis and tumor-size-dependent remission.

Overexpression of the receptor tyrosine kinase HER-2/neu is associated with poor prognosis in patients with breast and ovarian cancer. Recent excitement has surrounded the therapeutic effects of HER-2-blocking therapy strategies and has rekindled interest on the molecular mechanisms of HER-2/neu in tumor biology. To study the role of HER-2/neu overexpression in vivo, we used a murine fibroblast cell line (NIH3T3-her2) conditionally expressing human HER-2/neu under control of a tetracycline-responsive promoter. Expression of HER-2 could be down-regulated below detection limit (>625-fold dilution) by exposure of NIH3T3-her2 cells to anhydrotetracycline (ATc). Subcutaneous injection of NIH3T3-her2 cells into nude mice resulted in rapid tumor growth. Mice with mean tumor volumes of 0.2, 0.8, 1.9, and 14.9 cm(3) were treated daily with 10 mg/kg ATc to switch off HER-2/neu expression, producing reductions in tumor size of 100, 98.1, 81.4, and 74.2%, respectively, by 7 days after onset of ATc administration (P = 0.005, Kruskal-Wallis test). Different long-term effects of HER-2 down-regulation were observed when mice with small (0.2 cm(3); n = 7), intermediate (0.8-1.2 cm(3); n = 10) and large (> or =1.9 cm(3); n = 11) tumors received ATc for up to 40 days. Complete remission was observed for 100, 40, and 18% of the small-, intermediate-, and large-sized tumors, respectively (P = 0.003). However, after 20-45 days of ATc administration, recurrent tumor growth was observed for all mice, even in those with previous complete remissions. The time periods for which mean tumor volume could be suppressed to volumes <0.1 cm(3) under ATc administration were 34, 22, 8, and 0 days for tumors with initial volumes of 0.2, 0.8, 1.9 and 14.9 cm(3), respectively (P = 0.005, Kruskal-Wallis test). Interestingly, HER-2 remained below the detection limit in recurrent tumor tissue, suggesting that initially HER-2-dependent tumors switched to HER-2 independence. The "second hits" leading to HER-2-independent tumor growth have not yet been identified. The rapid regression of tumors after down-regulation of HER-2 was explained by two independent mechanisms: (a) a block in cell cycle progression, as evidenced by a decrease in Ki-67 antigen expression from 40% before ATc treatment to 8.3% after 7 days of ATc treatment; and (b) induction of apoptosis as demonstrated by caspase-3 activation and by the terminal deoxynucleotidyltransferase (Tdt)-mediated nick end labeling assay (TUNEL). In conclusion, we have shown that switching off HER-2 may disturb the sensitive balance between cell proliferation and cell death, leading to apoptosis and tumor remission. Tumor remission was dependent on the volume of the tumors before down-regulation of HER-2/neu.

CT attenuation of paired HRCT scans obtained at full inspiratory/expiratory position: comparison with pulmonary function tests.

The purpose of this prospective study was to measure lung attenuation at paired HRCT obtained at full inspiratory/expiratory position, to correlate with pulmonary function tests (PFTs) and to characterize different types of ventilatory impairment. One hundred fifty-five patients with and without pulmonary disease underwent paired HRCT obtained at full inspiratory/expiratory position. Three scan pairs were evaluated by densito- and planimetry using dedicated software. The PFTs were available for correlation in all patients (mean interval 5 days). Mean lung density (MLD) at full inspiration was -813 HU, and MLD at full expiration was -736 HU; both, as well as the expiratory attenuation increase, demonstrated significant correlations with static and dynamic lung volumes: up to r=0.68, p<0.05 for residual volume. The MLD and emphysema indices correlated markedly better for scans obtained at full expiration than at full inspiration, e.g. correlation with the residual volume: r=0.68 compared with r=0.55. Even better correlations were obtained for the lung area (229 cm(2) at inspiration, 190 cm(2) at expiration), up to r=0.74 for the lung area in expiration and the intrathoracic gas volume. Inspiratory MLD and the expiratory attenuation increase were able to differentiate obstructive and restrictive ventilatory impairment from normal subjects, the best results were obtained from scans obtained at full expiratory position ( p<0.05). In conclusion, scans obtained at full expiratory position reveal more functional information than scans obtained at full inspiratory position. Quantitative analysis of CT obtained at full expiratory position provides good estimations of static and dynamic lung volumes as well as significant differences between normal subjects and patients with ventilatory impairment.

Quantitative analysis of VEGF-isoforms in head and neck squamous cell carcinoma cell lines: relation to xenotransplantability and tumour progression in mice.

Overexpression of vascular endothelial growth factor (VEGF) is related to tumour progression and xenotransplant-ability in various human solid tumours, but the specific impact of the VEGF-subtypes is still under discussion. The aim of this study was to analyse a possible association of the major VEGF-isoforms and the growth characteristics of xenotransplanted human head and neck squamous cell carcinomas in nude mice. Seven SCC cell lines were analysed by quantitative RT-PCR using the TaqMan-System. We investigated the expression of VEGF-total-mRNA and of the major subtypes VEGF-121, -165, and -189 by using subtype specific primers. The cell lines were xenotransplanted in three mice each, and the data of tumour growth and progression were correlated to the expression of VEGF-isoforms. Six out of the seven cell lines analysed expressed all isoforms of VEGF in different quantities. One cell line expressed generally low levels of VEGF and no VEGF-189 at all. In this cell line xenotransplantation failed in one mice out of three. In a second cell line transplantation also failed in one out of seven mice. Success rates for the other five cell lines were 100%. The cell lines with higher transplantation success were expressed higher VEGF-121/165-189 ratios compared to those without success. In contrast, linearity of tumour growth and lack of necrosis were associated with a lower VEGF-121/165-189 ratio. The findings demonstrate a predominant expression of VEGF-165 and VEGF-189, compared to VEGF-121. We discuss an association of 'growth response rate' measured by tumour growth per detected VEGF-level. In highly proliferating tumours this rate appeared to be about 10 times higher than in low proliferating tumours. We conclude that the ratio between the VEGF-subtypes during tumour implantation and growth is a prerequisite for progression and hypothesise an individual and different response of each tumour cell line to VEGF.