BESTIMMUNG DER GENETISCHEN EMPFINDLICHKEIT FUR DIE ENTWICKLUNG VON PLATTENEPITHELKARZINOMEN DES KOPF-HALS-BEREICHS MIT BLEOMYCIN-INDUZIERTER CHROMOSOMALER INSTABILITAT BESTIMMUNG DER GENETISCHEN EMPFINLICHKEIT FUR DIE ENTWICKLUNG VON PLATTENEPITHELKARZINOMEN DES KOPF-HALS-BEREICHS MIT BLEOMYCIN-INDUZIERTER CHROMOSOMALER INSTABILITAT

Background: Cigarette smoking is a well established risk factor for head and neck squamous cell carcinoma. However, data on smoking history are not sufficient to predict which patients are at high risk for the development of multiple primary tumors. It is hypothesized that both exposure to carcinogens and individual susceptibility determine cancer risk. The aim of this study was to investigate whether a possible individual susceptibility for head and neck squamous cell carcinoma was related to the outcome of the mutagen sensitivity assay, which was adopted from Hsu and colleagues. Methods: Cultured peripheral blood lymphocytes were treated for five hours with 30 mlU/ml of bleomycin, and sensitivity was estimated by the mean number of chromatid breaks per cell scored in 100 metaphases of duplicate cultures. Results: It was shown that using this standard mutagen sensitivity assay, a person's sensitivity can be reproducibly measured. Age had a significant but small contribution, whereas gender and tobacco and alcohol use had no influence on the outcome of the assay. Therefore, we consider this sensitivity for the DNA damaging effect of bleomycin constitutional. A retrospective study showed that the breaks per cell in head and neck squamous cell carcinoma patients (0.96 ± 0.31; n = 52) were significantly higher (p<0.001) than in control persons (0.77 ± 0.19 n = 50). In addition, head and neck cancer patients who had already developed multiple primary tumors had a break per cell level (1.20 ± 0.47; n=20) which was even significantly higher (p<0.025) than in patients with one primary tumor. Conclusion: We hypothesize that mutagen sensitivity reflects an individual's sensitivity vor DNA damaging agents. The extremely high sensitivity in patients with multiple primary tumors suggests that mutagen sensitivity is a phenotype reflecting a person's susceptibility to head and neck cancer. We postulate that mutagen sensitivity in combination with smoking history can be used to identify those patients at highest risk for the development of multiple primary tumors

Comparative study of the sensitivity of head and neck cell lines to methotrexate (MTX) and the analog 10-ethyl, 10-deazaaminopterin (10-EdAM)

Squamous cell lines cultured in vitro provide a potential test system for the selection of analogs that have an improved therapeutic index. The growth inhibitory effects of methotrexate and the new folate analog 10-ethyl, 10-deazaaminopterin were compared in three in vitro-cultured human head and neck squamous cell carcinoma cell lines. The inhibitory concentrations of the new analog were 10- to 100-fold lower than the inhibitory concentrations of methotrexate. The sensitivity of these three head and neck squamous cell carcinoma cell lines to both drugs was essentially the same as the sensitivity of a rhabdomyosarcoma cell line known to be very sensitive to methotrexate when the cell line is grown as a xenograft in athymic nude mice. These data indicate that 10-ethyl, 10-deazaaminopterin may be a new and effective agent against head and neck squamous cell carcinoma

Plasma retinoid levels in head and neck cancer patients: A comparison with healthy controls and the effect of retinyl palmitate treatment

Vitamin A and related compounds, also known as retinoids are thought to play a role in the development of head and neck cancer. We measured levels of the major retinoids, retinol, all-trans retinoic acid, 13-cis retinoic acid and 13-cis-4-oxo retinoic acid in plasma of head and neck cancer patients in comparison with controls without cancer. No differences were found between plasma levels of these retinoids between 25 head and neck cancer patients and 21 controls. Mean baseline levels for the patients were 2458, 6.0, 6.4 and 8.6 nM for retinol, all-trans retinoic acid, 13-cis retinoic acid and 13- cis-4-oxo retinoic acid, respectively. In addition, we selected 10 patients from the chemoprevention trial Euroscan and measured the effect on retinoid levels of 300,000 I.U. daily retinyl palmitate intake during 1 month. Medication caused significant elevations in retinol levels (1.2 fold), all- trans retinoic acid (2.2 fold) and its metabolites 13-cis retinoic acid (5.8 fold) and 13-cis-4-oxo retinoic acid (8.9 fold). Because of its high increase in levels, 13-cis-4-oxo retinoic acid seems a good candidate to serve as a suitable marker to monitor patient compliance in future chemoprevention trials involving retinoids. No relations were found between the occurrence of side-effects of retinyl palmitate and retinoid levels during treatment. However, the two patients who developed side-effects had the highest pre- treatment levels of 13-cis retinoic acid and 13-cis-4-oxo retinoic acid, suggesting that retinoid toxicity is associated with relatively high basal retinoid metabolism

Enhanced turnover of all-trans-retinoic acid and increased formation of polar metabolites in head and neck squamous cell carcinoma lines compared with normal oral keratinocytes

Retinoids show promise in the treatment of various (pre)malignancies, including head and neck squamous cell carcinoma (HNSCC). Previous studies have shown that the metabolic pathways of retinoids are important in the anticancer effect of retinoids, and that these pathways may change during carcinogenesis. In the present study, we analyzed HNSCC cell lines (n = 11) and normal oral keratinocyte cultures (n = 11) by reverse-phase high-performance liquid chromatography and conducted growth inhibition assays. We demonstrate here that in contrast to normal oral keratinocytes, HNSCC cell lines: (a) had averaged a 17-fold greater turnover rate of all-trans-retinoic acid (RA); (b) had a 1.9-fold less RA-induced growth inhibition; (c) were able to form polar metabolites; and (d) were able to catabolize 4-oxo-RA. Furthermore, the mRNA expression of the RA-specific 4-hydroxylase, CYP26A1, was dramatically increased after RA-induction in the two HNSCC cell lines with the highest metabolism, was undetectable in normal keratinocytes, and was not inducible by RA. Next, introduction of CYP26A1 cDNA in a low-metabolizing HNSCC cell line resulted in an 11-fold higher turnover rate of RA and a 12-fold increase in the amount of polar metabolites, but it did not change sensitivity to RA. These observations point to fundamental changes in RA metabolism pathways during HNSCC carcinogenesis and may provide clues to a more rational approach for RA-mediated intervention

Lack of effect of daily N-acetylcysteine supplementation on mutagen sensitivity

The European Organization for Research and Treatment of Cancer multicenter Euroscan trial was set up to prevent the occurrence of second primary tumors in the upper aerodigestive and respiratory tract in patients cured for early stage head and neck squamous cell carcinoma. One randomized group of patients receive daily N-acetylcysteine, an antioxidant that may be protective especially in the early steps of carcinogenesis. Mutagen sensitivity, measured as sensitivity to bleomycin in peripheral blood lymphocytes, has been found to be increased in head and neck squamous cell carcinoma and is hypothesized to reflect cancer susceptibility. The aim of this study was to investigate whether mutagen sensitivity is influenced by oral N-acetylcysteine supplementation and can therefore be used as intermediate end point in chemoprevention. Patients (n = 19) who had various periods of N-acetylcysteine supplementation (600 mg daily for 3-9 months) were analyzed. In addition, a patient group (n = 14) that did not receive N- acetylcysteine supplementation was analyzed for comparison. Our results show no evidence that administration of N-acetylcysteine did influence the mutagen sensitivity level. The only explanatory variable in the analysis of the difference between two samples of one person was the b/c value of the first measurement. Moreover, the variability in these repeated measurements (coefficient of variation of 14%) indicates that additional studies should be performed to minimize this variability and to optimize the testing of mutagen sensitivity to accurately identify individual patients at high risk fur the development of multiple primary tumors

Enhanced turnover of all-trans-retinoic acid and increased formation of polar metabolites in head and neck squamous cell carcinoma lines compared with normal oral keratinocytes

Retinoids show promise in the treatment of various (pre)malignancies, including head and neck squamous cell carcinoma (HNSCC). Previous studies have shown that the metabolic pathways of retinoids are important in the anticancer effect of retinoids, and that these pathways may change during carcinogenesis. In the present study, we analyzed HNSCC cell lines (n = 11) and normal oral keratinocyte cultures (n = 11) by reverse-phase high-performance liquid chromatography and conducted growth inhibition assays. We demonstrate here that in contrast to normal oral keratinocytes, HNSCC cell lines: (a) had averaged a 17-fold greater turnover rate of all-trans-retinoic acid (RA); (b) had a 1.9-fold less RA-induced growth inhibition; (c) were able to form polar metabolites; and (d) were able to catabolize 4-oxo-RA. Furthermore, the mRNA expression of the RA-specific 4-hydroxylase, CYP26A1, was dramatically increased after RA-induction in the two HNSCC cell lines with the highest metabolism, was undetectable in normal keratinocytes, and was not inducible by RA. Next, introduction of CYP26A1 cDNA in a low-metabolizing HNSCC cell line resulted in an 11-fold higher turnover rate of RA and a 12-fold increase in the amount of polar metabolites, but it did not change sensitivity to RA. These observations point to fundamental changes in RA metabolism pathways during HNSCC carcinogenesis and may provide clues to a more rational approach for RA-mediated intervention

Expression of retinoic acid receptor gamma correlates with retinoic acid sensitivity and metabolism in head and neck squamous cell carcinoma cell lines

Retinoids, analogues of vitamin A, can reverse premalignant lesions and prevent second primary tumors in patients with head and neck squamous cell carcinoma (HNSCC). The effects of retinoids are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which act as ligand-activated transcription factors. The regulation of cell growth, differentiation and retinoid metabolism in normal, premalignant and malignant cells by retinoids is thought to be a result of their effects on gene expression. We investigated mRNA expression of RARs (α, β, and γ) and RXR-β by means of RNase protection and related this to retinoic acid (RA)-induced growth inhibition and RA turnover in four HNSCC cell lines (UM-SCC-14C, UM-SCC-22A, UM-SCC-35 and VU-SCC-OE). An RA-resistant subline of UM-SCC-35 was generated by exposure to increasing concentrations of RA for 8 months (designated UM-SCC-35R). RA turnover was determined on the basis of decreasing RA levels in the cells and culture medium after exposure to I μM RA. We found that RAR-γ mRNA expression was strongly correlated with RA-induced growth inhibition (p = 0.016, R = 0.92) and RA turnover (p = 0.041, R = 0.86). RAR-β transcript levels were reduced in three of five cell lines compared with normal mucosa, and these did not correlate with RA-induced growth inhibition and RA turnover. Expression of RAR-α and RXR-β was not substantially altered in any of the cell lines. These findings suggest that in HNSCC cell lines RAR-γ is the most important retinoid receptor for regulation of RA turnover rate and RA-induced growth inhibition

Considerations for in vitro retinoid experiments: Importance of protein interaction

Retinoids, natural and synthetic substances structurally related to vitamin A, are important modulators of cell proliferation and differentiation, and have proven activity in cancer therapy. Experiments to reveal the mechanism of action of retinoids are routinely performed in in vitro models. As retinoids are relatively hydrophobic and unstable, we hypothesized that the composition of culture media is of critical importance for the stability and bioavailability of these compounds. Various culture media were incubated with all-trans-, 13-cis- and 9-cis-retinoic acid (RA). Without fetal calf serum (FCS) or bovine serum albumin (BSA) in the medium, the concentration of these retinoids was found to decrease to considerably low levels. This excessive loss of retinoids was due to absorption to culture plates, reaction tubes and pipet tips. Binding of retinoids to BSA was demonstrated to have attenuating effects on uptake and metabolism of all-trans-RA, as studied in oral keratinocytes and head and neck cancer cells, indicating that a balance exists between the bioavailability and the aspecific loss of retinoids. In this study we demonstrate that the type of culture medium and especially the presence of protein in the medium is of paramount importance to perform reproducible experiments with retinoids. Copyright (C) 1999 Elsevier Science B.V