Irinotecan and radiation in vitro and in vivo

Loss of chromosome sequences at 13q14 (Rbl) and 17p13 (p53) associated with squamous cell carcinoma of head and neck (SCCHN) was evaluated in 12 recurrent tumors and 51 primary tumors from 63 patients. The incidence of LOH at 17p13 was 19 of 50 (38%) tumors, and at 13q14 was 21 of 57 (37%). LOH affecting Rbl and/or p53 was observed in 30 of 63 (48%) SCCHN. Coincident LOH at Rbl and p53 was detected in 10 of 46 (22%) tumors. There were nine cases in which primary and metastatic tumors were obtained from the same patient. Of these, seven were informative and five of these (71%) manifested LOH at p53 in both primary and metastatic sites. Examination of Rbl in these same tumors showed LOH in six of the nine metastases, and of these six, only three revealed LOH in the primary tumor. LOH at p53 or Rbl alone showed no correlation with clinical outcome. However, tumors that manifested LOH at both loci were associated with poorer patient outcome and poorer histological differentiation.     

TCP and NTCP in preclinical and clinical research in Europe

European research in radiation oncology has a long and successful tradition. The aim of this research is to increase the therapeutic window of radiotherapy by increasing the tumor control probability (TCP) and/or by decreasing the normal tissue complication probability (NTCP). This paper summarizes the basic radiobiological concept underlying treatment optimization by TCP-NTCP data and discusses some of the limitations of currently used models. These are controversial in many aspects and cannot be recommended for clinical routine practice but should rather be considered as a research tool.     

Daunorubicin and daunorubicinol pharmacokinetics in plasma and tissues in the rat

Recent evidence suggests that 13-hydroxy metabolites of anthracyclines may contribute to cardiotoxicity. This study was designed to determine the pharmacokinetics of daunorubicin and the 13-hydroxy metabolite daunorubicinol in plasma and tissues, including the heart. Fisher 344 rats received 5 mg kg^–1 daunorubicin i.v. by bolus injection. Rats were killed at selected intervals for up to 1 week after daunorubicin administration for determination of concentrations of daunorubicin and daunorubicinol in the plasma, heart, liver, kidney, lung, and skeletal muscle. Peak concentrations of daunorubicin were higher than those of daunorubicinol in the plasma (133±7 versus 36±2 ng ml^–1;P<0.05), heart (15.2±1.4 versus 3.4±0.4 g g^–1;P<0.05), and other tissues. However, the apparent elimination half-life of daunorubicinol was longer than that of daunorubicin in most tissues, including the plasma (23.1 versus 14.5 h) and heart (38.5 versus 19.3 h). In addition, areas under the concentration/time curves (AUC) obtained for daunorubicinol exceeded those found for daunorubicin in almost all tissues, with the ratios being 1.9 in plasma and 1.7 in the heart. The ratio of daunorubicinol to daunorubicin concentrations increased dramatically with time from <1 at up to 1 h to 87 at 168 h in cardiac tissue. Thus, following daunorubicin injection, cumulative exposure (AUC) to daunorubicinol was greater than that to daunorubicin in the plasma and heart. If daunorubicinol has equivalent or greater potency than daunorubicin in causing impairment of myocardial function, it may make an important contribution to the pathogenesis of cardiotoxicity.     

Advances in molecular diagnostics and therapeutics in head and neck cancer

Opinion statement Extensive treatment-related morbidities and stagnant survival rates over the past few decades for patients with squamous cell cancer of the head and neck (SCCHN) emphasize the need for novel diagnostics and therapeutics based on the molecular characteristics of the tumor. The development of an early detection test remains largely preliminary. Much attention has recently been given to saliva-based early detection assays that use accepted tumor markers such as p53 and DNA methylation. Most of these studies have focused on feasibility as opposed to prospective clinical trials. To date, early detection saliva assays have failed to yield a high enough sensitivity and specificity for broad population-based screening. The use of saliva as a noninvasive, inexpensive, and accessible diagnostic substrate remains desirable. Unlike SCCHN diagnostics, molecular-targeted therapies for SCCHN will soon be a reality, with many more compounds in the pipeline. The most promising of these drugs target the epidermal growth factor receptor (EGFR), which is known to be overexpressed in squamous cell carcinomas. Cetuximab, a monoclonal EGFR antibody, has shown efficacy in combination with radiotherapy in advanced SCCHN in a recent phase III trial and is currently being petitioned for US Food and Drug Administration approval. Likewise, erlotinib, an EGFR tyrosine kinase inhibitor, has shown favorable results in phase II trials as monotherapy and in combination with chemotherapy. Gefitinib, another EGFR tyrosine kinase inhibitor, has shown efficacy as monotherapy, in combination with chemotherapy, and with chemoradiotherapy. At least two phase III trials of gefitinib in patients with advanced SCCHN are ongoing. Such low-toxicity, tumor-specific targeting strategies will soon be available for patients with head and neck cancer. The challenge is to establish assays to determine which patients are most likely to benefit from these agents.     

Estrogen and Progesterone in Normal Mammary Gland Development and in Cancer

There is emerging evidence that the mammary epithelium in both mice and humans is arranged as a hierarchy that spans from stem cells to differentiated hormone-sensing, milk-producing and myoepithelial cells. It is well established that estrogen is an important mediator of mammary gland morphogenesis and exposure to this hormone is associated with increased breast cancer risk. Yet surprisingly, the primitive cells of the mammary epithelium do not express the estrogen receptor-α (ERα) or the progesterone receptor. This article will review the mammary epithelial cell hierarchy, possible cells of origin of different types of breast tumors, and the potential mechanisms on how estrogen and progesterone may influence the different subcomponents in normal development and in cancer. Also presented are some hypothetical scenarios on how this underlying biology may be reflected in the behavior of ERα(+) and ERα(-) breast tumors.     

The induction of tumours in the guinea-pig with methylcholanthrene and diethylnitrosamine and their propagation in vivo and in vitro

Tumour induction with diethylnitrosamine (DEN) and methylcholanthrene (MCA) has been studied in 3 strains of guinea-pig. A DEN concentration of 80 μg/ml drinking water daily proved too toxic but reasonable survival was obtained with 20 μg/ml 3 times per week in Hartley guinea-pigs and a local inbred strain. Heston Strain 13 guinea-pigs were particularly susceptible to the toxic effects of the diethylnitrosamine. In all3strains, 100% of the animals which survived the early toxic effects subsequently developed hepatomata, the mean time being 15 months. Methylcholanthrene was less toxic but more erratic as a carcinogen, the incidence of tumours in Hartley guinea-pigs varying from 18 to 100% in different experiments, the mean time of tumour development being 10 months.     

Transforming growth factor-beta and response to anticancer therapies in human liver and gastric tumors in vitro and in vivo

Liver cancer and gastric cancer are the most common solid tumors worldwide. Transforming growth factor-beta (TGF-beta) production and lack of response to TGF-beta growth inhibitory effects have been associated with tumor progression and therapeutic resistance. HepG2, Hep3B, and SK-HEP-1 human liver cancer lines produce 3, 5.7, and 2.5 ng TGF-beta1; 1.4, 2, and 4 ng TGF-beta2 and 0.15, 0.2 and 0.22 ng TGF-beta3 per 107 cells (24 h). Expression of the TGF-beta type I receptor is 20x, 1x, and 0.6x the level in mink lung MvLu1 cells in the HepG2, Hep3B, and SK-HEP-1 cells, respectively. HepG2 and Hep3B cells do not express the TGF-beta type II receptor while SK-HEP-1 cells express 7x the level found in mink lung MvLu1 cells. Hs 746T, KATO III, RF-1, and RF-48 human gastric cancer cell lines produce 12. 5, 0.35, 0.4, and 0.4 ng TGF-beta1; 2.6, 0.95, 0.5, and 0.52 ng TGF-beta2 and 0.42, 0.17, 0.12, and 0.14 ng TGF-beta3 per 107 cells (24 h). Expression of TGF-beta type I receptor is 0.7x, 0.7x, 0.8x, 0.6x the level in mink lung MvLu1 cells in the Hs 746T, KATO III, RF-1 and RF-48 cells, respectively. KATO III cells are lacking in the TGF-beta type II receptor while Hs 746T, RF-1 and RF-48 cells express 10x, 0.8x, and 1x the levels in mink lung MvLu1 cells. The IC50 for TGF-beta1 is >10 ng/ml in all of these lines except RF-48 where TGF-beta1 is mitogenic. The response of the cell lines to radiation, doxorubicin, mitomycin C, cisplatin, 5-fluorouracil, methotrexate, and gemcitabine showed that SK-HEP-1 was the most drug resistant liver cancer cell line and KATO III was the most drug resistant gastric cancer cell line. Overall, there was no correlation between TGF-beta secretion in cell culture and sensitivity of the cells to anticancer agents. Increased TGF-beta1 levels were detectable in the plasma of nude mice bearing Hep3B and Hs 746T xenografts. Those tumors which secreted greater amounts of TGF-beta were more therapeutically resistant in vivo.     

Catalysis of nitrosation in vitro and in vivo in rats by catechin and resorcinol and inhibition by chlorogenic acid

Measurements were made of the effects of phenolic compounds,some of which are present in the human diet, on the nitrosationof proline by nitrite to give N-nitrosoproline (NPRO). In vitro,resorcinol, catechin, p-nitrosophenol and phenol were catalystsand chlorogenic acid an inhibitor; guaiacol showed a marginalcatalytic effect. Both the catalytic and the inhibiting effectswere dependent on pH and on the concentration of phenolic compounds;catalysis by resorcinol and catechin was increased at optimalratios of [nitrite]: [phenolic compound]. Endogenous nitrosationwas examined in vivo by co-administration of nitrite, prolineand a phenolic compound to rats and by monitoring the amountof NPRO excreted in the urine. Under similar experimental conditions,the catalytic effects observed in vivo decreased in the sameorder as those observed in vitro: resorcinol > p-nitrosophenol> catechin > phenol guaiacol; chlorogenic acid acted asan inhibitor. Catalysis and inhibition of N-nitrosation in ratsin vivo appears to occur via mechanisms similar to those invitro, although the effects in vivo were smaller. The implicationsof our findings for the endogenous formation of N-nitroso compoundsand for variations in exposure due to different dietary constituentsin humans are discussed.     

Foscan and foslip based photodynamic therapy in osteosarcoma in vitro and in intratibial mouse models

Current osteosarcoma therapies cause severe treatment‐related side effects and chemoresistance, and have low success rates. Consequently, alternative treatment options are urgently needed. Photodynamic therapy (PDT) is a minimally invasive, local therapy with proven clinical efficacy for a variety of tumor types. PDT is cytotoxic, provokes anti‐vascular effects and stimulates tumor cell targeting mechanisms of the immune system and, consequently, has potential as a novel therapy for osteosarcoma patients. This study investigated the uptake and the dark‐ and phototoxicity and cytotoxic mechanisms of the photosensitizer (PS) 5,10,15,20‐tetrakis(meta‐hydroxyphenyl) chlorine (mTHPC, Foscan) and a liposomal mTHPC formulation (Foslip) in the human 143B and a mouse K7M2‐derived osteosaroma cell line (K7M2L2) in vitro. Second, the tumor‐ and metastasis‐suppressive efficacies of mTHPC formulations based PDT and associated mechanisms in intratibial, metastasizing osteosarcoma mouse models (143B/SCID and syngeneic K7M2L2/BALB/c) were studied. The uptake of Foscan and Foslip in vitro was time‐ and dose‐dependent and resulted in mTHPC and light dose‐dependent phototoxicity associated with apoptosis. In vivo, the uptake of both i.v. administered mTHPC formulations was higher in tumor than in healthy control tissue. PDT caused significant (Foscan p < 0.05, Foslip p < 0.001) tumor growth inhibition in both models. A significant (Foscan p < 0.001, Foslip p < 0.001) immune system‐dependent suppression of lung metastasis was only observed in the K7M2L2/BALB/c model and was associated with a marked infiltration of T‐lymphocytes at the primary tumor site. In conclusion, mTHPC‐based PDT is effective in clinically relevant experimental osteosarcoma and suppresses lung metastasis in immunocompetent mice with beneficial effects of the liposomal mTHPC formulation Foslip.     

Difference in the distribution of low-molecular-weight peptides and amino-acids in normal and malignant-cells and tissues

The amount of low molecular weight amino acids and peptides measured in the form amino acids released by hydrolysis was found to be very low in malignant tissues and cell lines compared to normal tissues and cell lines. The ratios of acid soluble proteins to these low molecular weight compounds were statistically different in normal and malignant cells. The difference may reflect faster growth, greater membrane permeability or other aspects of the malignant process itself.     

C-type virus particles in spontaneous and virus-induced leukemia and malignant lymphomas in mice and rats

The presence and numbers of C-type virus particles in an animal model consisting of mice and rats with either spontaneous or virus-induced leukemia and lymphomas were studied, in order to determine the relation of the appearance of virus particles to viral etiology of such neoplasms. The numbers and distribution of C-type virus particles in organs from 13 mice with spontaneous leukemia and lymphomas were compared with the presence of virus particles in organs of 13 mice with leukemia and lymphomas induced by passage A (Gross) virus inoculation. C-type virus particles were present in organs of all mice with either spontaneous leukemia or leukemia and lymphomas induced by virus inoculation. However, the number of particles observed was significantly higher in those mice in which leukemia was induced by virus inoculation. Virus particles were also observed, but in substantially smaller numbers, in organs of 13 of 25 untreated, healthy mice of the nonleukemic C3H(f) inbred line. In contrast to mice, C-type, or any other virus particles, were not found in organs of 10 Sprague-Dawley, Long-Evans or Sprague-Dawley X Long-Evans F1 hybrid rats with spontaneous leukemia. However, C-type virus particles were consistently present in organs of 11 Sprague-Dawley rats with leukemia induced by rat-adapted passage A (Gross) mouse leukemia inoculation. The virus particles appeared in the organs of the inoculated rats 5 days after i.p., and 11 days after s.c. inoculation. Virus particles were not found in organs of 10 healthy untreated Sprague-Dawley rats. The implications of these observations are discussed.     

Stage and survival in breast cancer patients in screened and non-screened Danish and Swedish populations

Comparisons between the Danish and Swedish Cancer Registry revealed a 9% difference in 5-year survival for breast cancer patients diagnosed between 1983 and 1989. The purpose of this study was to determine whether previous differences in survival and stage still exist or whether the Danish figures approach those of the Swedish registry. Complete population-based cohorts of patients with breast cancer in the years 1996-1997 from well-defined areas in Denmark and Sweden were compared. The study regions were a Danish (Funen) and a Swedish (Malm?) county with mammography screening and two Danish counties without screening. No difference in extent of disease or survival was observed between screening areas regardless of country. However, there were significant differences in stage distribution and survival between screening populations and Danish non-screening populations, to the benefit of populations provided with a screening programme. Five-year survival was 5-6%, higher in screening populations than in Danish non-screening counties. Corresponding disease-specific survival enhanced the difference. In a multivariate analysis increasing age, tumour size and stage decreased survival. Adjusting for these factors eliminated differences in survival according to country/county. Survival difference could be attributed to early diagnosis and favourable stage in populations offered mammography screening and was not related to nation. Denmark may, through early detection, approach the beneficial stage distribution and survival observed in Sweden.     

Comparative analysis of integrins in vitro and in vivo in uveal and cutaneous melanomas.

Changes in integrin expression have been shown to be important for the growth and metastatic capacity of melanoma cells. In this study, we have examined the expression of alphav integrins by three uveal and four cutaneous malanoma lines. No lines expressed alphavbeta6 and only TXM13, a cutaneous line, expressed alphavbeta8. All lines expressed alphavbeta5 and alphavbeta3 (four out of four cutaneous, two out of three uveal) or avpl (OM431, an uveal line). Thus, OM431 is the second uveal melanoma we have described that expresses alphavbeta1 and this, we report again, functions as an alternative vitronectin/fibronectin receptor. Subcutaneous growth of cell lines in athymic mice correlated with an alphavbeta3-positive, alphavbeta1 -negative phenotype. Analysis of clinical material from cutaneous melanoma showed that although alphav expression was increased in 88% of metastases, this could not all be explained by up-regulation of alphavbeta3, with only 2 out of eight skin metastases expressing this heterodimer. Using antibody SZ.21, which as we report here works in archival material, only 1 out of 15 uveal metastases expressed detectable beta3. Thus, acquisition of alphavbeta3 expression, which has been implicated in cutaneous melanoma progression, may not be required for development of metastases from uveal melanoma or indeed for skin, as distinct from lymph node, metastases of cutaneous melanoma.     

Conventional cytogenetics and fluorescence in situ hybridization in persistent cytopenias and myelodysplastic syndromes in childhood

Accurate detection of the abnormal clone in children with persistent cytopenia (PC) may confirm the diagnosis of myelodysplastic syndrome (MDS) and determine prognosis and evolution of the disease. Bone marrow (BM) samples were obtained from 65 children, 11 of which were finally diagnosed as primary or secondary MDS. Ten to 20 G-banded metaphases were analyzed and FISH was performed using a-satellite probes for chromosomes 7 and 8. Conventional cytogenetic analysis (CCA) was successful in 40/65 samples, revealing clonal aberrations in 3 patients with MDS. FISH was successful in all cases, detecting monosomy 7 and trisomy 8 abnormal clones in 5 patients. Abnormalities were identified in 3/6 children with primary MDS and 3/5 with secondary MDS. None of the patients with PC of etiology other than MDS had a clonal abnormality in the BM. The results confirm the high incidence of chromosome abnormalities in childhood MDS and the sensitivity of FISH in detecting minor abnormal clones.     

Modifications in phospholipase D activity and isoform expression occur upon maturation and differentiation in vivo and in vitro in human myeloid cells.

Activation of phospholipase D (PLD) occurs in response to various stimuli and results from the activity of two isozymes, hPLD1 and hPLD2. PLD activity appears to be involved in several myeloid cell processes during their development and activation, including proliferation of myeloblasts in the bone marrow and secretion, phagocytosis and NADPH oxidase activation, essential functions of differentiated neutrophils. The present work studies PLD characteristics, activity and both isozyme expression during maturation and differentiation of myeloid cells by using three different systems: leukemic myeloblasts at different stages of maturation, terminally differentiated neutrophils ex vivo and four human myeloid cell lines, NB4, HL-60, PLB 985 and U937, induced to differentiate with alltrans retinoic acid (ATRA), a cyclic adenosine monophosphate (cAMP) analogue or both agents together. HL-60, a bipotential cell line has also been differentiated along the granulocytic pathway with DMSO and the monocytic pathway with 1,25-dihydroxy vitamin D3. In all these systems, PLD activity increases with maturation and differentiation whatever the inducer used and the granulocytic or monocytic pathways. Increase in basal activity which reflects the expression during development of both hPLD1 and hPLD2 appears to be mainly related to the former isozyme expression. Association of PLD characteristic changes with maturation and differentiation was also confirmed using two NB4 clones resistant to these processes. Comparison between PLD characteristics in myeloblasts during maturation and differentiation ex vivo and in vitro in the different cell lines demonstrated that NB4 induced to differentiate with ATRA represents the best model for further studies on the specific roles of each PLD isoform in various functions of differentiated myeloid cells.