Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

MMP-9 gene ablation mitigates hyperhomocystenemia-induced cognition and hearing dysfunction

Hyperhomocysteinemia (HHcy) is associated with cognitive decline and hearing loss due to vascular dysfunction. Although we have shown that HHcy-induced increased expression of matrix metalloproteinase-9 (MMP-9) is associated with cochlear pathology in cystathionine-β-synthase heterozygous (CBS^+/?) mice, it is still unclear whether MMP-9 contributes to functional deficit in cognition and hearing. Therefore, we hypothesize that HHcy-induced MMP-9 activation causes vascular, cerebral and cochlear remodeling resulting in diminished cognition and hearing. Wildtype (WT), CBS^+/?, MMP-9^?/? and CBS^+/?/MMP-9^?/? double knock-out (DKO) mice were genotyped and used. Doppler flowmetry of internal carotid artery (ICA) was performed for peak systolic velocity [PSV], pulsatility index [PI] and resistive index [RI]. Cognitive functions were assessed by Novel Object Recognition Test (NORT) and for cochlear function Auditory brainstem response (ABR) was elicited. Peak systolic velocity, pulsatility and resistive indices of ICA were decreased in CBS^+/? mice, indicating reduced perfusion. ABR threshold was increased and maximum ABR amplitude and NORT indices (recognition, discrimination) were decreased in CBS^+/? mice compared to WT and MMP-9^?/?. All these parameters were attenuated in DKO mice suggesting a significant role of MMP-9 in HHcy-induced vascular, neural and cochlear pathophysiology. Regression analysis of PSV with ABR and cognitive parameters revealed significant correlation (0.44–0.58). For the first time, MMP-9 has been correlated directly to functional deficits of brain and cochlea, and found to have a significant role. Our data suggests a dual pathology of HHcy occurring due to a decrease in blood supply (vasculo-neural and vasculo-cochlear) and direct tissue remodeling.     

In vitro development of angiosperm floral buds and organs

In the last twenty-five years, young inflorescences, floral buds and individual floral organs of a number of species have been cultured in vitro. There is considerable variability in the requirement of plant growth regulators and nutritional factors for flower development of different species. This variability is compounded by the fact that the hormonal and nutritional requirements are different at various stages of organ and floral development. Experimental studies on normal and mutant flowers in vitro have provided insights into some of the regulatory processes in floral organogenesis. The potential use of the in vitro technique in elucidating the various mechanisms in flower development is stressed.     

Evidence that the capsule around mycobacteria grown in axenic media contains mycobacterial antigens: implications at the level of cell envelope architecture

The intracellular growth of pathogenic mycobacteria has been linked to the presence of an electron transparent zone (ETZ or capsule), which surrounds the phagocytized bacteria and prevents the diffusion of lysosomal enzymes in infected macrophages. Recently, it was suggested that this capsule may be a bacterial structures, even being present in test tube-grown pathogenic mycobacteria (FEMS Microbiol. Lett. 1988, 56, 225-230). In the present paper, we show that under special fixation and embedding conditions, this capsule was clearly observed among 7 strains of mycobacteria grown in axenic media and also in M. leprae extracted and purified from experimentally infected armadillo or nude mice. In the case of bacteria treated likewise but subject to a prior dehydration step, this capsular structure disappeared suggesting its lipidic nature. Ultrathin sections of M. intracellular after immunolabelling showed for the first time that this capsule obtained mycobacterial antigens confirming its mycobacterial origin. It is suggested that the mycobacterial capsule may be formed of inert lipids, in which surface antigens are embedded.     

Electron cytochemistry of mycobacteria: Evidence that strongly acidic sulfate groups are present on the surface of H37Rv (virulent) strain ofMycobacterium tuberculosis

Cytochemical characterization of mycobacterial surfaces was carried out on virulent (H37Rv) and avirulent (H37Ra) strains ofMycobacterium tuberculosis. The results were quantified and compared with those obtained with three colony types of the opportunistic pathogenMycobacterium avium. Mycobacterium aurum, a rapidly growing, nonpathogenic species, served as a model for the cytochemical methods. Concanavalin A (ConA) reacted with -d-mannose and -d-glucose residues, whereas negative charged residues were detected with either the ionized ferritin (CF) or the colloidal ferric hydroxide (CIH) method. Strongly acidic sulfate groups were detected by their selective blockage with alcian blue (AB) at pH 1 prior to the CIH labeling at pH 1.8. Weakly acidic groups were demonstrated by AB blockage at pH 2.5 prior to staining with CF stain. Except forM. aurum, all other strains showed a marked heterogeneity in regard to the abundance of their surface labeling. Accessible sulfate groups were present on the cell surface of the virulent H37Rv strain ofM. tuberculosis, but not on the avirulent strain H37Ra. Distribution of ConA receptors, on the other hand, was unrelated to the virulence or pathogenicity of the bacterial strain.     

Spatial and temporal organization of actin during hydration,activation, and germination of pollen inPyrus communis L.: a population study

Summary Dynamics of F-actin organization during activation and germination ofPyrus communis (pear) pollen was examined using rhodaminephalloidin. Prior to activation, the rhodamine-phalloidin labelling pattern appeared as circular profiles in the peripheral cytoplasm of the vegetative cell and as coarse granules around the vegetative nucleus. In activated pollen, parallel arrays of cortical F-actin were aligned circumferentially, along the polar axis in non-apertural areas of the pollen grain, and at 45° to 90° to the polar axis beneath the apertures. Some pollen also showed fluorescent granules or fusiform bodies dispersed throughout the cytoplasm, but as the number of such pollen diminished with prolonged incubation, these are being considered as intermediate patterns. In later stages, the filaments became organized as interapertural bundles traversing the three apertures. However, prior to emergence of the pollen tube, labelling became confined to a single aperture. In germinated pollen grains, actin microfilaments are aligned more or less axially with respect to the axis of the developing pollen tube.The granular labelling pattern seen around the vegetative nucleus prior to pollen activation also became clearly filamentous with pollen activation; this filamentous pattern persisted until germination when it was replaced by cables that aligned longitudinally with respect to the emerging tube axis.The results demonstrate that the organization of actin undergoes considerable changes in the period preceding pollen germination and that microfilament polarization is achieved before pollen germination.     

Long-term low-dose mutation studies in human cells: metanil yellow and orange II

We tested the mutagenic effects of two commonly used fold colors, metanil yellow and orange II, in AHH-1 human lymphoblast cells. The cell line, which is competent for oxidative metabolism of various chemicals, was exposed to both compounds in high-dose x short-term (3 day) or high-dose x long-term (10-day) and low-dose x long-term (20-day) treatments. Concentrations of metanil yellow and orange II as low as 22 nM and 12 nM, respectively, were sufficient to induce mutation rates which were equal to twice the spontaneous mutation rate at the HPRT locus in AHH-1 cells.