Different breakage-prone regions on chromosome 1 detected in t(11;14)-positive mantle cell lymphoma cell lines and multiple myeloma cell lines are associated with different tumor progression-related mechanisms.

To better define secondary aberrations that occur in addition to translocation t(11;14)(q13;q32) in mantle cell lymphomas (MCL) and in multiple myelomas (MM), seven t(11;14)-positive MCL cell lines and four t(11;14)-positive MM cell lines were analysed by fluorescence R-banding and spectral karyotyping (SKY). Compared with published data obtained by G-banding, most chromosome aberrations were redefined or further specified. Furthermore, several additional chromosome aberrations were identified. Thus, these cytogenetically well defined t(11;14)-positive MCL and MM cell lines may be useful tools for the identification and characterization of genes that might be involved in the pathogenesis of MCL and MM, respectively. Since MCL and MM were found to have different alterations of chromosome 1, these were investigated in more detail by fluorescence in situ hybridization (FISH) and multicolor banding (MCB) analyses. The most frequently altered and deletion-prone loci in MCL cell lines were regions 1p31 and 1p21. In contrast, breakpoints in MM cell lines most often involved the heterochromatic regions 1p12-->p11, and the subcentromeric regions 1q12 and 1q21. These data are in accordance with previously published data of primary lymphomas. Our findings may indicate that different pathways of clonal evolution are involved in these morphologically distinct lymphomas harboring an identical primary chromosome aberration, t(11;14).

Malignant transformation and viral replication of rat bone and muscle cells after in vitro infection with rat-adapted murine sarcoma virus (Moloney).

Isolated bone and muscle cells from rat fetuses were infected with the rat-adapted osteosarcomogenic murine sarcoma virus (Moloney) for examination of malignant transformation and viral replication. After the infection, the bone cells underwent an unusual transformation characterized by two patients in the focus formation. In the early transformation (Transformation I), foci consisting of only morphologically altered cells appeared, but they soon disappeared. Focus formation of this type continued for subsequent cell passages. In the late transformation (Transformation II), typical foci of malignant cells were formed with rapid multiplication. The cells in both types of transformation produced sarcomogenic as well as leukemogenic viruses. They contained C-type particles showing varying morphology and dimensions. Distinct bone cell tropism of osteosarcomogenic murine sarcoma virus (Moloney) was shown for three in vitro passages through rat bone cells. The infected muscles cells produced Transformation I foci at an incidence lower than did the bone cells, being mostly nontransformed. The virus yielded from these cells was predominantly leukemogenic, consisting of typical C-type particles. Evidence presented suggests that the tissue dependency with replicating ability of murine sarcoma virus and murine leukemia virus present in osteosarcomogenic murine sarcoma virus (Moloney) preparation is responsible for exhibiting the tissue tropism of this virus.

Predisposition of cloned fetal hamster lung epithelial cells to transformation by a precarcinogen, benzo(a)pyrene, using growth hormone supplementation and collagen gel substratum.

A cloned fetal Syrian hamster lung epithelial cell line (M3E3/C3) was used to compare the influence of two different culture conditions on the degree of cellular differentiation and susceptibility of the cells to undergo malignant transformation by a precarcinogen, benzo(a)pyrene. Conventional conditions consisted of growth medium containing Roswell Park Memorial Institute Medium 1640, pyruvate, and fetal bovine serum and a substratum of plastic. Complex conditions comprised the growth medium supplemented with insulin, hydrocortisone, estradiol, epidermal growth factor, transferrin, and cholera toxin and a substratum of collagen gel. Under the complex culture conditions, there was extensive development of endoplasmic reticulum and Golgi vesicles, whereas under conventional conditions these organelles were only minimally developed. This was correlated with 1.5-1.8 times enhancement of ethoxycoumarin deethylase and reduced nicotinamide adenine dinucleotide phosphate-dependent cytochrome c reductase activities. Decomposition of added benz(a)anthracene into water-soluble compounds increased with the period of incubation and reached about 40% of initial benz(a)anthracene (50 micrograms/10 ml/flask) at 48 h under the complex conditions, whereas under the conventional conditions only less than 4% decomposition occurred. Benzo(a)pyrene in the dose range 2-8 micrograms/ml was strongly cytotoxic and caused significant anchorage independent transformation only under complex culture conditions. Transformed cells produced tumors in two of four hamsters during 8 months following s.c. injection within 48 h of birth. These results suggest that the complex culture conditions predisposed the cloned fetal epithelial cells to malignant transformation by benzo(a)pyrene through stimulation of cellular differentiation and development of enzyme systems capable of activating it metabolically.

Failure to transmit diethylnitrosamine tumorigenicity from transplacentally exposed F1 generation Syrian hamsters to the respiratory tract of F2 and F3 generations.

A multigeneration study with four successive generations of Syrian hamsters was conducted to determine whether a single s.c. injection of different doses of diethylnitrosamine (DEN) (1.25, 2.5, 5, 10, and 20 mg/kg body weight) on day 15 of pregnancy induces respiratory tract tumors not only in the treated P generation mothers and their F1 progeny but also in F2 and F3 generations. In this study, the P generation mothers only were given a single injection of DEN during the period of gestation. Fifty-six % of the 36 DEN-treated mothers and 52% of their F1 generation offspring (total, 233 animals) developed neoplasms in the respiratory tract. A single respiratory tract tumor was found in one DEN-unexposed F1 generation control hamster as well as in one F2 generation animal (total, 209 animals) descended from DEN-exposed P generation. Both tumors were considered to have arisen spontaneously. No respiratory tract tumors were observed in the F3 generation (total, 160 animals) descended from a DEN-exposed P generation. Thus our results indicate that the vertical transmission of the tumorigenic effect of DEN in Syrian hamsters is limited to one generation and does not persist in the F2 and F3 generations.

Challenge for 3D culture technology: Application in carcinogenesis studies with human airway epithelial cells.

Lung cancer is still one of the major intractable diseases and we urgently need more efficient preventive and curative measures. Recent molecular studies have provided strong evidence that allows us to believe that classically well-known early airway lesions such as hyperplasia, metaplasia, dysplasia and carcinoma in situ are really precancerous lesions progressing toward cancer but not necessarily transient and reversible alteration. This suggests that adequate early control of the precancerous lesions may lead to improved prevention of lung cancer. This knowledge is encouraging in view of the imminent necessity for additional experimental systems to investigate the causal mechanisms of cancers directly in human cells and tissues. There are many questions with regard to various precancerous lesions of the airways. For example, should cells, before reaching a stage of invasive carcinoma, undergo all precancerous stages such as hyperplasia or metaplasia and dysplasia, or is there any shortcut to bypass one or more of the precancerous stages? For the study of such questions, the emerging 3-dimensional (3D) cell culture technology appears to provide an effective and valuable tool. Though a great challenge, it is expected that this in vitro technology will be rapidly and reliably improved to enable the cultures to be maintained in an in vivo-mimicking state of differentiation for much longer than a period of at best a few months, as is currently the case. With the help of a "causes recombination-Lox" (Cre-lox) technology, it has been possible to trace cells giving rise to specific lung tumor types. In this short review we have attempted to assess the future role of 3D technology in the study of lung carcinogenesis.

Importance of coronary collaterals for restoration of left ventricular function after intracoronary thrombolysis.

In an evaluation of the role of coronary collaterals in the early period of acute myocardial infarction (AMI), 30 patients with acute total coronary occlusion treated with intracoronary thrombolysis 2 to 8 hours after the onset of symptoms were studied. Only 13 patients with well-developed collaterals in the early period of AMI and successful thrombolysis showed improvement of global and regional ejection fraction (EF) from the acute phase to the chronic phase (global EF from 50% to 71%, p less than 0.001; regional EF from 25.4% to 49.2%, p less than 0.001). In patients with no or less well-developed collaterals and successful thrombolysis, global and regional EF were similar to those in patients in whom thrombolysis was unsuccessful. Among the 19 patients with successful thrombolysis, there was no significant correlation between the duration of ischemia and the improvement of regional EF (r = -0.03, difference not significant). These data suggest that the extent of coronary collateral vessels in the early period of AMI is an important determinant of restoration of left ventricular function after intracoronary thrombolysis.

Transformation-dependent quantitative changes in glycopeptide binding to concanavalin A-sepharose.

Differentially L-fucose-labelled glycopeptides from the surface of a Syrian golden hamster (SGH) fetal lung control cell line were compared with those from chemically-transformed and tumour cell lines derived from the control line by cochromatography on Concanavalin A-Sepharose (Con A-Sepharose) and Sephadex G-50. Quantitative differences were found both in the unbound and specifically-bound fractions between control and transformed cells upon Con A-Sepharose chromatography. In the glycopeptides from transformed and tumour cells, the unretarded fraction was concomitantly decreased compared to the controls. When the ratio of unbound to specifically-bound fractions was used, a statistically significant difference could be calculated between the values of control versus transformed or tumour cells. In all transformed and tumour cell lines investigated, the quantitative change in Concanavalin A binding, expressed as an increase of the ratio of unretarded to specifically-bound glycopeptides, was paralleled by a shift of transformed or tumour glycopeptides to higher apparent molecular weight compared to the control in gel chromatography.

Toxicity and chromosomal damage in fetal Syrian hamster and human pulmonary epithelial cells.

In order to test the hypothesis that human chromosomes are more stable than those of rodents, we compared the fetal human and Syrian hamster pulmonary epithelial cell lines for their sensitivity to the induction of cytotoxicity (CT), chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs). One day after plating, the cells were exposed for 2 h to various doses of ENU dissolved in an exposure medium consisting of McIlvaine buffer, RPMI 1640, and bovine serum albumin. CAs and SCEs were examined in 24-64 post-exposure hours by the standard methods, while CT was determined by cell counts. The frequency of CAs (particularly chromatid exchanges) and that of SCEs showed clear dose dependency and was remarkably higher in the hamster than in the human cells. CT determined in 1 week of post-exposure incubation showed a similar dose response for both cell lines with sharply declining regressions. These results suggest that human chromosomes are indeed more resistant to ENU-inducible aberrations than hamster chromosomes and that CT may not always be directly associated with chromosomal damage.

Unscheduled DNA synthesis and HPRT mutation in fetal Syrian hamster and human respiratory epithelial cells exposed to ethylnitrosourea.

We previously reported that the chromosomes of fetal Syrian hamster respiratory epithelial cells were less stable toward ethylnitrosourea (ENU) than those of comparable human cells. Following this, we compared the sensitivity of genetic materials of the same cell systems to the same mutagen in terms of unscheduled DNA synthesis (UDS) and mutation at HPRT locus (HPRT-). UDS occurred 5 (with 0.1 mg ENU/ml) to 7 (with 0.4 mg ENU/ml) times more frequently in the hamster cells than in the human cells. This much lower UDS frequency in human cells cannot be solely explained by the fact that the human cells possess only a moderately larger (1.6 to 2.9 times) size of intracellular deoxythymidine triphosphate (dTTP) pool than the hamster cells. This finding would thus indicate that the hamster cells actually carry out DNA repair, whether correct or aberrant, more often than the human cells. Moreover, HPRT- was also 5 (at 0.4 mg/ml) to 26 (at 0.8 mg/ml) times more frequent in the hamster cells than in the human cells. Therefore, the current results suggest that the DNA repair mechanisms of the hamster cells are less accurate and more unstable than those of the human cells. Our previous findings with regard to the chromosomal stability give support to this hypothesis.

Epithelial alterations in fetal tracheal explants of Syrian golden hamsters exposed to diethylnitrosamine in utero.

The fetal tracheae (13th--15th day of gestation) of Syrian golden hamsters exposed in utero to diethylnitrosamine (DEN: 200--400mg/kg body wt.) were divided into cranial and caudal halves. Both sections were then organ-cultured for 4--6 weeks. The cranial explants developed 1.5 times as many epithelial tracheal lining alterations as the caudal explants. These changes were found 25--42 days after the beginning of explant culture and histologically demonstrated hyperplasia, metaplasia and dysplasia. No alterations occurred in the control explants.

Toxic and transforming effects of polycyclic hydrocarbons in fetal hamster lung-cell cultures. I. Benzo(alpha)pyrene.

When seeded at a low cell density in Petri dishes not pre-conditioned with feeder layers, fetal hamster lung cells at 2-4 passages grew with greatly varied plating efficiencies (PE). Addition of insulin to the serum-supplemented medium gave relatively constant PE. However, the presence of insulin in the medium enhanced the toxicity of benzo(alpha)pyrene (BP) on these cells. Cytotoxic and cell transforming effects of BP in the presence of insulin were studied.

The possibilities of reproducing effects of benzo[a]pyrene in a transformation test using Syrian hamster fetal lung cells.

Various dose levels of benzo[a]pyrene (BP) were tested on short-term cultured foetal hamster lung cells for transforming effects in vitro in 6 similar experiments. The statistical evaluation of the results indicated a weak reproducibility (P = 0.09). Factors which inherently influence such reproducibility were considered.

Comparison of chrysene metabolism in epithelial human bronchial and Syrian hamster lung cells.

Chrysene is metabolized to 1-, 2-, 3-, and 4-hydroxychrysene and trans-1,2- as well as trans-3,4-dihydroxydihydrochrysene in human and Syrian hamster epithelial lung cells as indicated by GC/MS analysis, whereas K-region oxidation is at most a very minor pathway. Cells of a permanent clonal line of fetal hamster lung metabolized 97% of the chrysene whereas fetal human bronchial epithelial cells converted 24% of the substrate within 8 days incubation. In human cells oxidation at the 3,4-position predominates, whereas oxidation at the 1,2-position is the major pathway in hamster cells. Indication for a bay-region oxidation of chrysene in hamster cells has been obtained.

New functional cell-culture approach to pulmonary carcinogenesis and toxicology.

Modern pulmonary toxicology (including lung carcinogenesis) has, to assist its rapid development, constantly incorporated the knowledge obtained through cell and tissue-culture studies. While this has been carried out in rather a passive manner until quite recently, the currently necessary multi-disciplinary approach increasingly requires more active involvement of cell/tissue-culture techniques in this area. Our understanding in this regard is that one of such requirements is to establish a cell-culture system consisting of a single population of possible target cells for certain classes of hazardous inhalants. In addition, such target cells in culture should be able to function in a manner as closely resembling the situation in vivo as possible. In view of the culture techniques presently available, this requirement is probably too ideal to be met immediately. Nevertheless, efforts have been made in the last decade to achieve functioning cultures of Clara cells, type II pneumocytes or small mucus granule cells (SMGC), using undifferentiated cells obtained from animal and human fetuses. This attempt forms a sharp contrast to the usual approach, in that while the latter tries to keep the functions of adult cells in an already differentiated state, the former aims at inducing functional differentiation in undifferentiated cells by manipulating culture conditions. In carrying out these efforts, we have shown clear evidence that the type II pneumocytes and Clara cells induced in vitro are closely cognate and share a common precursor cell in culture, and that SMGC are at a pre-stage of differentiation to Clara cells. We have also shown an induced capacity for xenobiotic activation and conjugation in SMGC in culture. Our next plan is to prove similar activity (of mixed-function oxidase) in Clara cells and type II pneumocytes induced to differentiate in culture.

A fetal respiratory epithelial cell line for studying some problems of transplacental carcinogenesis in Syrian golden hamsters.

Using repeated cloning and treatment with cis-HPL (200 micrograms/ml), an analogue of a procollagen precursor inhibitory to the growth of collagen-synthesizing cells of mesenchymal origin, clonally premature epithelial cell lines were isolated from fetal SGH lungs cultured on the 15th day of gestation. One of the cell lines, M3E3/C3, which has been extensively studied for biological characterization, developed poorly differentiated carcinomas in injected hamsters after transformation by MNNG. Moreover, when grown on collagen gel, this cell line indicated an obvious potency for in vitro differentiation in response to vitamin A by developing activated Golgi regions, well developed rER and a number of mucus-like granules. Since such a differentiative responses is expected to be definable in the light of respiratory epithelium developing in utero, this cell line may be useful for studying mechanisms of differentiation-dependent sensitivity of fetal organs to transplacental carcinogen exposure.

Respiratory sensitization to diphenyl-methane-4,4'-diisocyanate (MDI) in guinea pigs: impact of particle size on induction and elicitation of response.

The impact of particle size of aerosolized polymeric diphenylmethane-4,4'-diisocyanate (MDI) for the induction and elicitation of respiratory sensitization was evaluated. Four groups of 16 female guinea pigs each received either the vehicle, repeated intradermal (id) injections (3 x 0.3% MDI), one high-level inhalation exposure of 15 min to 135 mg MDI/m(3) air using a small aerosol (MMAD approximately 1.7 microm) or large aerosol (MMAD approximately 3.8 microm). Three weeks later, animals were challenged subsequently with two ramped concentrations of MDI aerosol (average concentrations 16 and 49 mg/m(3) air, each for 15 min) and two different particle sizes, i.e., the MMAD was either approximately 1.6 microm or approximately 5.1 microm for the small- and large-size aerosol, respectively. Respiratory sensitization was assessed by two endpoints: the measurement of respiratory rate, and examination of influx of eosinophilic granulocytes into the mucosa and submucosa of the trachea, bronchi, and lung-associated lymph nodes (LALN). The recruitment of eosinophilic granulocytes into bronchial tissues was subdivided as follows: muscularis mucosae, submucosa, and perivascular. From measurements of respiratory rate, it would appear that guinea pigs sensitized by id injections or by inhalation exposure with the large aerosol tended to display a higher responsiveness than naive controls when challenged with the small aerosol. The recruitment of eosinophilic granulocytes in the bronchial tissue was greater in both inhalation induction groups as compared to the vehicle control. It appears that there was a somewhat greater response in animals sensitized by id injections or by inhalation exposure with the large aerosol and challenged with the small aerosol. Topographically, this difference was apparent only at the bronchial perivascular level and lung-associated lymph nodes (LALN), whereas at the submucosal and muscularis mucosae level the impact on particle size tended to be less pronounced. In summary, this study suggests that a brief, high-level inhalation exposure of MDI aerosol caused a sensitization of bronchial tissues in guinea pigs. The higher sensitization potency of the large aerosol may possibly be related to a dosimetric phenomenon because of the greater fraction of deposition of large particles within the upper respiratory tract. Overall, challenge exposures with this type of irritant aerosol appear to evoke more consistent effects when the MMAD is in the range of approximately 2 rather than approximately 5 microm.

Molecular cytogenetic characterization of the mantle cell lymphoma cell line GRANTA-519.

Combining fluorescence R-banding, fluorescence in situ hybridization and spectral karyotyping allowed us to precisely define chromosomal breakpoints, gains, losses and a newly detected amplification in the human mantle cell lymphoma (MCL) cell line GRANTA-519. GRANTA-519 is characterized by the t(11;14)(q13;q32) resulting in overexpression of cyclin D1, a key player in cell cycle control. Hitherto unresolved complex rearrangements involve 1p, 1q, 3cen, 9p, 11q, 12p, 12q, 16p, 17p, and 18cen. Moreover, a 4- to 6-fold gain of sequences on 18q leads to a low-level amplification of the BCL2 gene and to an overexpression of the BCL2 protein. These results provide the basis for the identification of not only candidate oncogenes responsible for MCL in gained regions, but also for the identification of putative tumor suppressor genes in commonly deleted regions like 1p22, which would eventually enable functional studies of these genes.

Sensitivity of Syrian golden hamster fetal lung cells to benzo[a]pyrene and other polycyclic hydrocarbons in vitro.

Dose responses were compared of cultured fetal Syrian golden hamster lung cells (FSHL) to the toxic and transforming effects of benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[B]F), benz[a]anthracene (B[a]A, indeno[1,2,3-c,d]pyrene (I[c,d]P), benzo[k]fluoranthene (B[k]F) and benzo[e]pyrene (B[e]P). Effort was first given to standardising the techniques for evaluating B[a]P dose-responses. These polycyclic aromatic hydrocarbons (PAH) were then tested at concentrations of up to 1 microgram/ml, and only B[a]P showed clear cytotoxicity. The transforming effects of B[b]F, B[a]A and I[c,d]P at 1 microgram/ml appeared comparable to those of B[a]P at 0.05 microgram/ml.

Chromosomal aberrations in tracheal epithelial cells of the fetal Syrian golden hamster after transplacental N-diethylnitrosamine administration.

Transplacental effect of N-diethylnitrosamine (DEN) on chromosomal morphology, was investigated in fetal tracheal epithelium of Syrian hamsters. At the 15th day of pregnancy, Syrian golden hamsters were injected subcutaneously with a tumorigenic dose of DEN (50, 100 and 200 mg/kg body wt). Two hours later, the fetal tracheae were isolated, epithelial cells of the respiratory mucosa were separated from mesenchymal tissue and were transferred to cell culture. At 24, 48 and 72 h after cultivation, chromosomal damages were examined. The results showed clearly that a high incidence of chromosomal aberrations, particularly chromatid-type exchanges, were seen in the epithelial cells from DEN-treated groups.

In vitro reconstituted tissue as an alternative to human respiratory tract.

The data obtained from in vitro systems utilizing human cells and tissues should form a basic part of the information necessary for risk assessment. The most important thing for such systems is, therefore, to simulate the structures and functions of cells and tissues in the native organ as closely as possible. In designing in vitro systems, there may be two approaches-one aiming at the growth of cells in a primarily two-dimensional fashion, the other allowing cells to form in vivo-mimicking three-dimensional architectures. In cultures in which the airway epithelial cells are growing in a two-dimensional fashion, some functional and structural characteristics can be developed to a considerable extent. However, there are some that cannot be developed or expressed under that condition but require a three-dimensional growth pattern. In this paper we explore the capacity of early to long-term passage airway epithelial cells (human and hamster) to resume architectures and functions existing in the native tissue in the specific environments given in vitro.