SR 4233 (tirapazamine): a new anticancer drug exploiting hypoxia in solid tumours

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide, WIN 59075, tirapazamine) is the lead compound in a new class of bioreductive anticancer drugs, the benzotriazine di-N-oxides. It is currently undergoing Phase I clinical testing. The preferential tumour cell killing of SR 4233 is a result of its high specific toxicity to cells at low oxygen tensions. Such hypoxic cells are a common feature of solid tumours, but not normal tissues, and are resistant to cancer therapies including radiation and some anticancer drugs. The killing of these tumour cells by SR 4233, particularly when given on multiple occasions, can increase total tumour cell killing by fractionated irradiation by several orders of magnitude without increasing toxicity to surrounding normal tissues. Topics covered in this review include the rationale for developing a hypoxic cytotoxic agent, the cytotoxicity of SR 4233 as a function of oxygen concentration, the mechanism of action of the drug and its intracellular target and the in vivo evidence that the drug may be useful as an adjunct both to radiotherapy and chemotherapy. Finally, the major unanswered questions on the drug are outlined.     

Variable expression of Mn SOD in three different human melanoma cell lines

The mechanism of age-related cortical bone loss was investigated in 229 Japanese women, 41-94 years of age, by metacarpal bone mass measurement. While no significant correlation was found between bone width and age, a significant increase in bone marrow width, and significant decreases in cortical bone density and total bone mass were observed in association with aging (P < 0.0001). There was a significant negative correlation between total bone mass and bone marrow width (r = -0.239; P < 0.0005), and significant positive correlations between both total bone mass and cortical bone density (r = 0.539; P < 0.0001) and cortical bone width (r = 0.839; P < 0.0001). The findings suggested that age-related cortical bone loss in middle-aged and elderly women resulted from two different factors; a decrease in cortical bone density caused by progression of intracortical porosity, and a decrease in cortical bone width as a result of bone loss on the endosteal surface. The latter had a greater influence on an age-related cortical bone loss than the former.     

HNK-1 antigens on uveal and cutaneous melanoma cell lines

The HNK-1 epitope has been associated with the metastatic behaviour of uveal melanomas. We characterized HNK-1 antigens on four human uveal (primary and metastatic) and two primary cutaneous melanoma cell lines by immunocytochemistry and Western blot analysis. We also determined the involvement of the HNK-1 epitope in cell-cell interactions on a matrigel layer. Three uveal melanoma cell lines (one primary and two metastatic) and one cutaneous melanoma cell line showed HNK-1 expression by immunocytochemistry. On matrigel, only the HNK-1-positive cutaneous melanoma cell line Bowes grew in a honeycomb-like structure which disappeared after adding HNK-1 antibodies to the culture medium. Immunoblot analysis of the primary uveal melanoma cell line EOM-3 revealed five HNK-1-positive protein bands with apparent molecular weights of 200, 160, 115, 95 and 75 kDa. The cutaneous melanoma cell line Bowes showed three HNK-1-positive protein bands with apparent molecular weights of 150, 135 and 90 kDa. This study shows that two uveal (primary and metastatic) and one primary cutaneous melanoma cell lines express HNK-1 antigens on immunoblot. Only in the HNK-1-positive cutaneous melanoma cell line Bowes did the HNK-1 epitope have a function in intercellular adhesion. Although the primary uveal melanoma cell line EOM-3 showed a similar HNK-1 immunoreactivity, we could not demonstrate HNK-1-mediated cell adhesion. On immunoblot, the two cell lines displayed different HNK-1 antigens, which may explain the difference in cell adhesion.     

Establishment and characterization of three cell lines from Aedes triseriatus (Diptera: Culicidae)

The objectives of this study were to develop sex-, age-, and body size-specific nomograms and partition values for upper and lower limits of M-mode echocardiographic aortic root measurements derived from a large population-based cohort. The study sample consisted of 1433 male and 1816 female participants in the Framingham Heart Study and Framingham Offspring Study who were normotensive and free of clinically apparent heart disease at the baseline examination. Aortic root measurements were obtained by M-mode echocardiography by a leading-edge to leading-edge technique. The relations of age and measures of body size with aortic root dimensions were evaluated with sex-specific correlations and multiple stepwise linear regression analyses. Age was the most important determinant of aortic root size in both men and women in the multivariable regression models. Models with age and body surface area yielded R2 values of 0.214 in men and 0.222 in women. Models with age and height yielded lower R2 values of 0.136 in men and 0.181 in women. Thus aortic root dimensions vary widely with the age, sex, and body size of individuals. Sex-specific reference nomograms of aortic root dimensions in relation to age and body size (body surface area or height) are presented to facilitate the detection of abnormalities of aortic root size.     

Establishment and characterization of human ocular melanoma cell lines

Five continuous cell lines have been established from 29 ocular melanomas and maintained for periods ranging from 3 to 9 years in medium identical to that in which 3 concomitantly studied lines of cutaneous melanoma cells were cultured as controls. The long-term problems to be overcome in establishing uveal cell lines are related to cell-doubling times which ranged from 72 to 432 hr, and plating efficiency, which ranged from 0.5%-6.5%. Tumors and cell lines were found to contain melanosomes. The morphology of uveal cells during the early subcultures exhibited multiple changes. Two different established cell lines were obtained from one ciliary-body tumor. Biochemical studies revealed markers of melanogenesis and neuroendocrine compounds. Cytogenetic studies revealed chromosomal abnormalities that differed between uveal and conjunctival melanomas.     

三种缝线材料对人肺腺癌细胞A549增殖和细胞周期的影响

Objective: The interaction of cell and medical biomaterial is one of the significant factors to affect clinical application of medical biomaterial. This research is to investigate three of suture lines how to affect the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro. Methods: Three of suture lines were respectively cultivated with lung adenocarcinoma cell A549, after of 72 hours, we detected absorptions of each group by MTT method in order to reflect the proliferation of lung adenocarcinoma cell A549, and also examined percentage of G1 period cells and S period cells of each group by flow cytometry. Results: Different of suture lines had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P ＜ 0.05). The effect of absorbent suture line was the strongest on the proliferation and cell cycle of lung adenocarcinoma cell A549, the effect of chorda serica chirurgicalis was medium, and the effect of slide wire was poor. Different length of each suture line had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P ＜ 0.05).Conclusion: Three of suture line materials have different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549, with dose-effect relationship.     

Activation status and function of the VLA-4 (alpha4beta1) integrin expressed on human melanoma cell lines

A highly sensitive microspectrophotometer was developed to measure spectral changes of oxyhemoglobin (oxy Hb) in single red blood cells (RBC) incubated with stimulated macrophages as a model of nitric oxide (NO) dependent cytotoxicity. Our microspectrophotometer, using a modified acousto-optic tunable filter (AOTF) and a 2-dimensional CCD array, allows fast spectrophotometric data acquisition. Human RBC treated with various concentrations of NO showed spectral changes due to the conversion of oxy Hb to methemoglobin (met Hb), in which the change in absorption differences at alpha (557 590 nm) and beta (542-525 nm) bands showed a linear relationship with the concentration of NO up to 100 microM. In contrast to highly diffusible NO, nitrite ions (NO2-) seem to enter RBC very slowly, resulting in negligible formation of met Hb in the presence of 5 mM glucose even during a prolonged incubation period. RBC were incubated with murine macrophages with and without lipopolysaccharide (LPS) in the presence of glucose for 24 and 40 h and subjected to the microspectrophotometric assay. The RBC incubated with LPS-stimulated macrophages showed significant changes in the spectrum due to NO-dependent conversion of oxy Hb to met Hb, which corresponded to the spectral changes of RBC treated with a several times higher concentration of NO than that in the culture medium. The trapping efficiency was calculated from the amounts of the NO released from macrophages and of the met Hb formed in the RBC, which gave a high efficiency (43%). The results suggest that RBC trap NO directly by cell cell interaction with macrophages. This spectrophotometric system is available for use with just a few drops of samples to study NO-specific cytotoxicity as a model of RBC without the use of any chemical reagent, in parallel with microscopic observations on changes of the cellular morphology under physiological conditions, such as membrane damage leading to hemolysis, adherence, and phagocytosis.     

Suppression of tumorigenic and metastatic potentials of human melanoma cell lines by mutated (143 Val-Ala) p53

Metastatic melanoma, compared with other cancers, appears to be unusual because of its low frequency of p53 mutations and prevalence of wild-type p53 protein in advanced malignancy. Here, we examined the effects of wild-type and mutated p53 (143 Val-Ala) on tumorigenic and metastatic potential of two human melanoma cell lines. The cell line UISO-MEL-4 contains wild-type p53 and is tumorigenic, whereas UISO-MEL-6 lacks p53 and produces lung and liver metastasis upon s.c. injection into athymic mice. Our study showed that UISO-MEL-4 stably transfected with wild-type p53 cDNA driven by cytomegalovirus promoter-enhancer sequences expressed high levels of p53 and p21 and formed s.c. tumours in vivo. Mutated p53 (143 Val-Ala) expression, on the other hand, inhibited tumour growth in 50% of cases and produced significantly slower growing non-metastatic tumours. Reduced tumour growth involved necrotic as well as apoptotic cell death. Inhibition of tumour growth was abrogated by the addition of Matrigel (15 mg ml(-1)). With UISO-MEL-6 cells, stably transfected with mutant p53, tumour growth was delayed and metastasis was inhibited. In soft agar colony formation assay, both wild-type and mutant p53 transfectants reduced anchorage-independent colony formation in vitro. These data suggest that mutated (143 Val-Ala) p53, which retains DNA binding and some of the transactivation functions of the wild-type p53 protein, suppresses tumorigenic and metastatic potentials of human melanoma cell lines in vivo.     

Differential effects of interleukin 1-alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) on motility of human melanoma cell lines on fibronectin

Interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) induce a motogenic response in a number of benign and malignant cells. We examined the chemokinetic effects of these cytokines on the cell migration of four melanoma cell lines on fibronectin using modified Boyden chambers and video-time lapse analysis. Flow cytometry analysis of IL-1 receptors, TNF receptors, and shifts in beta 1 integrin expression were correlated with the effects of these cytokines on cell migration on fibronectin. The four melanoma cell lines exhibited heterogeneous expression of types I and II IL-1 receptors as well as p60 TNF receptors. Scant p80 TNF receptor expression was detected on only one cell line. Three of four melanoma cell lines demonstrated type I IL-1 receptors by Western blotting. IL-1 alpha and TNF-alpha induced heterogeneous modulation of beta 1 integrin expression in the four melanoma cell lines tested; downward shift of the alpha 2, alpha 3, alpha 4, and beta 1 integrin subunits was detected among three of the melanoma cell lines as were upward shifts of the alpha 4, alpha 5, and alpha 6 integrin subunits among three of the melanoma cell lines. IL-1 alpha and TNF-alpha induced enhanced migration on fibronectin in one of the melanoma cell lines and were related to an upward shift in the alpha 4 and alpha 5 integrin subunit expression. Taken together, the findings indicate that expression of a particular receptor for IL-1 or TNF does not necessarily signal a motogenic response in melanoma cells, but induces heterogeneous shifts in beta 1 integrin expression. However, upregulation in alpha 4 and alpha 5 integrin subunits appears to relate to enhanced migration on fibronectin.     

Analysis of p53 in human cutaneous melanoma cell lines

Mutations in the p53 tumour suppressor gene have been detected in a variety of human malignancies. Mutations have been found predominantly in conserved regions two to five. Our aim was to analyse p53 at the protein and DNA level in seven melanoma cell lines of cutaneous origin (HMB-2, DX3, LT5.1, MJM, SK23, A375P and A375M), including two parental/metastatic derivatives (A375P and A375M; DX3 and LT5.1). By immunohistochemical staining with three mouse monoclonal antibodies and a rabbit polyclonal serum, it was possible to observe differential nuclear expression of p53. The quantitation of p53 protein levels by ELISA correlated with the nuclear staining pattern. Western blotting showed an intact p53 protein in all cell lines; p53 was polymorphic in three cell lines (MJM, A375P and A375M). DNA sequencing studies showed that all cell lines had wild type p53. These results suggest that p53 is unlikely to play a significant role in the genesis of cutaneous melanoma.     

MEMD, a new cell adhesion molecule in metastasizing human melanoma cell lines, is identical to ALCAM (activated leukocyte cell adhesion molecule)

From a differential mRNA display comparing a non- and a highly metastasizing human melanoma cell line, we isolated and characterized memD. memD is preferentially expressed in the highly metastasizing melanoma cell lines of a larger panel. The encoded protein, MEMD, is identical to activated leukocyte cell adhesion molecule (ALCAM), a recently identified ligand of CD6. ALCAM is involved in homophylic (ALCAM-ALCAM) and heterophylic (ALCAM-CD6) cell adhesion interactions. We have studied MEMD/ALCAM cell-cell interactions between human melanoma cells. The expression of this cell adhesion molecule not only correlates with enhanced metastatic properties and with aggregational behavior of human melanoma cells as tested by FACS analysis, but transfection experiments also make clear that MEMD/ALCAM expression is essential for cell-cell interaction of the investigated human melanoma cells. As the melanoma cell lines analyzed are all CD6 negative, these results strongly suggest that MEMD/ALCAM is an adhesion molecule mediating homophylic clustering of melanoma cells. MEMD/ALCAM expression is not restricted to subsets of leukocytes and melanoma cells, it can also be found in healthy organs and in several other malignant tumor cell lines. Besides, MEMD/ALCAM is also expressed in cultured endothelial cells, pericytes and melanocytes, in xenografts derived from the radial and vertical growth phase and in 4 of 13 melanoma metastasis lesions. The potential role is discussed of MEMD/ALCAM mediated cell-cell interactions in migration of mobile cells (ie, activated leukocytes, metastasizing tumor cells) through tissues.     

Effect of dexamethasone on cell proliferation of neuroepithelial tumor cell lines

The effect of glucocorticoid on cell proliferation, the expression of glucocorticoid receptor, and the relationship between inhibition of cell growth and apoptosis were investigated in four established neuroepithelial tumor cell lines (KNS42, T98G, A172, and U251MG). Glucocorticoid receptor expression was located in the cytoplasm of untreated cells, but translocated into nuclei after treatment with dexamethasone in KNS42, T98G, and A172 cells. U251MG did not express glucocorticoid receptors. Dexamethasone significantly inhibited the growth of KNS42 and T98G cell lines, at high concentrations in contrast to growth stimulation at low concentration. Dexamethasone inhibited proliferation of A172 cell line at all concentrations from 10(-4) M to 10(-7) M. These were prevented by RU38486, a specific glucocorticoid antagonist. Apoptosis did not occur in any cell lines after dexamethasone treatment. There was no response to glucocorticoid by U251MG cells. Dexamethasone treatment of neuroepithelial tumor cells expressing glucocorticoid receptors causes translocation into the nucleus to modulate cell proliferation upon binding of different concentrations of dexamethasone in vitro. Dexamethasone inhibits proliferation of some neuroepithelial cell lines, not by glucocorticoid-induced apoptosis. The bimodal potential of glucocorticoid to stimulate or suppress proliferation of neuroepithelial tumor cells expressing glucocorticoid receptor must be considered in clinical trials.     

Hypermutability of UV-treated plasmids in dysplastic nevus/familial melanoma cell lines

Members of cutaneous melanoma (CM) families with dysplastic nevi (DN) are at high risk of developing CM. Using a shuttle vector plasmid, pSP189, cell lines from three patients with CM plus DN were previously found to have elevated post-UV plasmid mutability. To investigate familial occurrence of this cellular phenotype, we examined post-UV plasmid mutability in 31 lymphoblastoid cell lines from 6 familial CM kindreds. In comparison to 16 normal control lines, we found an abnormally elevated post-UV plasmid mutability in cell lines from 13 of 13 patients with CM plus DN (P = 1.5 x 10(-8)) and from 5 of 8 patients with DN only (P = 0.001). Elevated spontaneous plasmid mutation frequency (MF) was also present in cell lines from six of the CM plus DN patients (P = 0.002) and three of the DN-only patients (P = 0.028). However, cell lines from two patients with CM without DN had normal post-UV plasmid MF. Although not specific for CM patients, of 27 cell lines with elevated post-UV plasmid MF, only 8 were from donors who did not have CM + DN or DN (19 of 24 versus 8 of 28; P = 0.0003). This study indicates that post-UV plasmid hypermutability is a laboratory marker for members of melanoma-prone families and suggests that patients with familial CM have a defective mechanism for handling UV-induced DNA damage.     

Identification of PTEN/MMAC1 alterations in uncultured melanomas and melanoma cell lines

A novel tumor suppressor gene, PTEN/MMAC1, has been recently shown to be mutated in gliomas, breast, prostate, kidney cancers and melanomas. Loss-of-heterozygosity studies in melanoma have suggested the presence of at least one chromosome 10q locus lost early in tumor progression. In this study, we screened 45 melanoma cell lines and 17 paired uncultured metastatic melanoma and peripheral blood specimens for PTEN/ MMAC1 alterations using PCR-SSCP and direct sequencing. We found nine melanoma cell lines with homozygous deletions (five with intragenic loss) and four cell lines with mutations (one nonsense and one frameshift; two intronic); from among our uncultured melanoma specimens, we found one tumor with a somatic 17 bp duplication in exon 7 leading to a premature stop codon and one tumor with a possible homozygous deletion. Furthermore, we have identified a novel intragenic polymorphism within intron 4 of PTEN/MMAC1. Taken together, these data suggest that PTEN/MMAC1 may be a chromosome 10q tumor suppressor important in melanoma tumor formation or progression.     

Effect of sodium butyrate on human breast cancer cell lines

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 mM. The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G2M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.     

A synthetic peptide inhibitor for alpha-chemokines inhibits the growth of melanoma cell lines

Melanoma growth stimulatory activity (MGSA/GROalpha) is a 73 amino acid peptide sharing sequence characteristics with the alpha-chemokine superfamily. MGSA/GROalpha is produced by diverse melanoma cell lines and reported to act as an autocrine growth factor for the cells. We tested the binding of MGSA/GROalpha to melanoma cell lines, Hs 294T and RPMI7951, and found that these cells could bind to MGSA/GROalpha but not to interleukin-8. Recently, we defined a novel hexapeptide, antileukinate, which is a potent inhibitor of binding of alpha-chemokines to their receptors on neutrophils. When antileukinate was added to melanoma cells, it inhibited the binding of MGSA/ GROalpha. The growth of cells from both melanoma cell lines was suppressed completely in the presence of 100 microM peptide. The cell growth inhibition was reversed by the removal of the peptide from the culture media or by the addition of the excess amount of MGSA/GROalpha. The viability of Hs 294T cells in the presence of 100 microM peptide was > 92%. These findings suggest that MGSA/GROalpha is an essential autostimulatory growth factor for melanoma cells and antileukinate inhibits their growth by preventing MGSA/GROalpha from binding to its receptors.     

Effects of diheptyldiselenide (DDS) on human tumor cell lines and on peripheral blood mononuclear cells

In vitro effects of graded concentrations of diheptyldiselenide (DDS) on human tumor cell proliferation, and on the proliferative responses and immunological functions of peripheral blood mononuclear cells (MNC) were investigated. The agent significantly decreased tumor cell proliferation in a dose and time dependent manner. Proliferative responses of MNC to phytohemagglutinin (PHA) and interleukin-2 (IL-2) were also significantly depressed when MNCs were exposed to DDS (250 microM for 18 h) led to a significant increase in NK activity only in MNC samples showing very limited baseline NK function. On the other hand, generation of LAK cells was significantly inhibited by DDS. However, when the agent was added to the effector and target cell mixture during the 4 h 51Cr release cytotoxicity assay, no influence was found on NK and LAK-mediated target cell lysis. These studies show that high concentrations of DDS inhibit tumor cell proliferation and could also impair certain proliferative-dependent immune functions, without directly affecting cell-mediated cytolytic activity of effector cells.     

Effect of ionizing radiation on cell-cycle progression and cyclin B1 expression in human melanoma cells

In the present study we investigated the effect of gamma-irradiation (2.5 and 10 Gy) on cell-cycle progression of a human melanoma cell line, M14, characterized by a moderate radiosensitivity (SF2 = O.5). Flow cytometric analysis showed a dose-dependent S-phase accumulation, which was detectable 8 hr after treatment with 2 and 5 Gy and was still persistent at 12 hr after 10 Gy exposure. Such a delay in S-phase was paralleled or followed by an accumulation of cells in G2M, which was transient at the lowest radiation doses and still persistent at 72 hr after 10 Gy. Such an accumulation was, at least in part, due to a block in G2-M transition, as demonstrated by mitotic index analysis. Bivariate flow cytometric analysis of DNA content and cyclin B1 expression showed that, following 2 and 5 Gy, the fraction of cyclin B1-expressing cells was superimposable upon that of G2M cells. Conversely, in cells treated with 10 Gy, the fraction of cyclin B1-expressing cells was half the G2M cell fraction. Northern-blot analysis indicated that the radiation-induced decrease in cyclin B1 protein expression was accompanied by a reduced cyclin B mRNA level. On the whole, our results indicate a direct inhibitory effect of 10 Gy irradiation on cyclin B1 expression as a possible cause for the persistent G2 block in irradiated M14 cells.