Requirement for continuous antigenic stimulation in the development and differentiation of antibody-forming cells. The effect of passive antibody on the primary and secondary response.

The essential role of continuous antigenic stimulation in the development and differentiation of antibody-forming cells as defined in the X-Y-Z immune cell maturation scheme was examined in these studies. Mice were primed with sheep erythrocytes (SRBC) in an attempt to induce maximum immune progenitor cell conversion (X --> Y). Subsequently antigen was depleted at 1 or 4 days after priming with isologous specific antibody in order to interrupt further immune cell differentiation (Y --> Z). It was reasoned that this condition would result in depression of the functional antibody-producing cell compartment as measured in the intact mice and subsequently in enhancement of the sensitized (Y cell) compartment as measured in the spleen cell transfer system. These data were also correlated with systematic studies of the hyperplasia of the spleen germinal centers. The effect of passive antibody on the primary response to SRBC was a marked decrease indirect and indirect hemolysin-producing cells (DPFC and IPFC). However, there was a lack of correlation in the degree of antibody-mediated 19S and 7S immune cell suppression during the primary response, the DPFC being much less depressed than the IPFC. As measured in the transfer system there was an enhanced 19S sensitized cell compartment and a depressed 7S sensitized cell compartment in 1 day passively immunized mice. This was true whether or not transfers were performed 1, 2, or 4 wk after priming. Similarly, there was an enhanced 19S-sensitized cell compartment with little or no effect on the 7S-sensitized. cell compartment in 4 day passively immunized mice. These data suggest that progeny of the antigen-stimulated progenitor cells (X cell), as a consequence of lack of further antigenic stimulation, were forced into maturation arrest. These studies further demonstrate that isologous passive antibody suppresses germinal center growth regardless of whether the antibody is infused 1, 2, or 4 days after priming. In terms of formation of sensitized cells, the marked depression of 7S sensitized cell compartment after passive immunization at 24 hr in contrast to the enhancement of the 19S sensitized cell compartment corresponds to the suppressed growth of germinal centers during the primary response. Thus, if the germinal center is, as suggested, the site of proliferative expansion of immunocompetent cells, these data indicate that the germinal center growth is related to the 7S antibody response and in the formation of "7S memory."

Serum alpha globulin fraction: survival-and-recovery effect in irradiated mice.

An alpha macroglobulin fraction (19S) was isolated from the serum of rats and BC(3)F(1) mice by zonal ultracentrifugation. Both the isologous and heterologous macroglobulin fractions increased survival among BC(3)F(1) mice x-irradiated with 750 roentgens. The mouse macroglobulin fraction also enhanced radiation recovery of hematopoietic tissue as measured by colony-forming assay and iron-59 incorporation into erythropoietic cells. The overall difference in hematopoietic activity in the irradiated (400 roentgens) mice treated with the macroglobulin fraction, in comparison with this activity in the controls, was three- to fivefold in the bone marrow and nine- to tenfold in the spleen between days 4 and 7 after irradiation. This effect was not obtained with the isologous serum protein fraction containing proteins of smaller molecular weight.

Identification of seven rat axonemal dynein heavy chain genes: expression during ciliated cell differentiation.

Axonemal dyneins are molecular motors that drive the beating of cilia and flagella. We report here the identification and partial cloning of seven unique axonemal dynein heavy chains from rat tracheal epithelial (RTE) cells. Combinations of axonemal-specific and degenerate primers to conserved regions around the catalytic site of dynein heavy chains were used to obtain cDNA fragments of rat dynein heavy chains. Southern analysis indicates that these are single copy genes, with one possible exception, and Northern analysis of RNA from RTE cells shows a transcript of approximately 15 kb for each gene. Expression of these genes was restricted to tissues containing axonemes (trachea, testis, and brain). A time course analysis during ciliated cell differentiation of RTE cells in culture demonstrated that the expression of axonemal dynein heavy chains correlated with the development of ciliated cells, while cytoplasmic dynein heavy chain expression remained constant. In addition, factors that regulate the development of ciliated cells in culture regulated the expression of axonemal dynein heavy chains in a parallel fashion. These are the first mammalian dynein heavy chain genes shown to be expressed specifically in axonemal tissues. Identification of the mechanisms that regulate the cell-specific expression of these axonemal dynein heavy chains will further our understanding of the process of ciliated cell differentiation.

Tumor development in organ transplants obtained from carcinogen-exposed rats.

F344 rats were exposed intragastrically to two different dose levels of N-nitrosoheptamethyleneimine (NHMI) resulting in a cumulative dose of either 225 or 450 mg/kg body weight. Tumor development was followed either in situ in the NHMI-exposed animals or in tracheas and esophagi from NHMI-exposed donors after these organs were grafted to isogeneic recipients. Tumor responses in situ and in organ grafts were compared. The results showed that the process of carcinogenesis is not disrupted by the transplantation procedure. The carcinogen dose-response relationship observed in situ was also seen in the transplanted organs. At the high carcinogen dose, the tumor incidence was 100% in situ and transplanted esophagi and 20% in tracheas in situ compared to 25% in tracheal transplants. At the low dose, the tumor incidence was 36% in the esophagi in situ compared to 100% in transplanted esophagi, which suggests a greater sensitivity of the transplant system to detect the carcinogenicity of NHMI. The proportion of carcinomas to papillomas was markedly higher in transplanted esophagi. The tracheal tumor response at both NHMI dose levels showed the same trend but was too low to allow any firm conclusions.

Two-stage carcinogenesis studies with asbestos in Fischer 344 rats.

The cocarcinogenic effect of chrysotile asbestos was investigated in heterotopic tracheal transplants of F344 rats. Tracheal transplants were first exposed to graded doses of dimethylbenz[a]anthracene (DMBA) contained in intraluminal pellets. The doses ranged from 12.5 to 100 micrograms. Control tracheas received blank pellets. Four weeks after the start of DMBA exposure (when all carcinogen had been released from the pellets), the spent pellets were removed, and 200 micrograms chrysotile, a nontumorigenic dose of asbestos, was introduced into the lumina of the preexposed tracheas. No significant enhancement of the tumor response was seen with 100 micrograms DMBA, a dose that was tumorigenic by itself. However, with 50 and 25 micrograms DMBA, nontumorigenic dose levels, a 15 and 23% incidence of tracheal carcinomas occurred when DMBA exposure was followed by a nontumorigenic dose of chrysotile. At 12.5 micrograms DMBA, this effect was not observed. Whatever the mechanisms were that formed the basis of this tumor enhancement effect of asbestos, the consequences were similar to those observed with tumor promotion in classical two-stage carcinogenesis studies.

Unstable cellular differentiation in adenosquamous cell carcinoma.

For the determination of whether two or more stem lines with fixed of stable differentiation are present in a growing mixed adenosquamous cell carcinoma, two different mixed tumors, derived from transformed F344 rat tracheal epithelium, were dissociated and ten single cells were isolated from each tumor. Each of these single cells was allowed to grow 27 population doublings to form a clonal population. All clones were then inoculated into animals for the assessment of in vivo differentiation. All cell inocula produced carcinomas within 7-70 days. Of the ten single cell clones isolated from the first tumor, seven clones produced mixed adenosquamous carcinomas and three produced adenocarcinomas in which no squamous cell component was found. From the second tumor, eight clones produced mixed tumors and two produced squamous cell carcinomas in which no adenocarcinoma component was found. The mixed tumors derived form clones contained widely varying proportions of adeno and squamous components. Recloning of the cloned lines yielded populations that produced mixed adenosquamous cell carcinomas upon inoculation in vivo. Comparison of tumor phenotypes from clones of early and late passages further confirmed the instability of differentiation in these neoplastic epithelial cells. These studies strongly suggest that the mixture of differentiated cell types in adenosquamous cell carcinomas is not the result of a mixture of stem cells with one fixed phenotype within the tumor, but rather is the result of an instability of differentiation; the neoplastic cells can express both types of differentiation.

Carcinogenic and cocarcinogenic effects of inhaled synthetic smog and ferric oxide particles.

The carcinogenic and cocarcinogenic activity of synthetic smog, ferric oxide (Fe2O3) dust, and a mixture of the two air contaminants was determined in a long-term inhalation study with Syrian hamsters. Inhaled Fe2O3 particles definitely enhanced diethylnitrosamine tumorigenicity in the peripheral lung. Synthetic smog did not. When tested at a concentration of 40 ppm methane equivalents or 40 mg/m3, respectively, neither air pollutant by itself appeared carcinogenic. Fe2O3 caused pulmonary fibrosis and synthetic smog caused alveolar bronchiolization in many of the exposed animals.

Epithelial focus assay for early detection of carcinogen-altered cells in various organs of rats exposed in situ to N-nitrosoheptamethyleneimine.

The purpose of these studies was to determine a) whether epithelial cells with altered in vitro growth capacity occur not only after topical application of 7-12-dimethylbenz [a]-anthracene but also after systemic administration of a carcinogenic nitrosamine, and b) whether such cells can be isolated from tissues other than tracheal mucosa. AT 3 and 20 weeks following intragastric administration of 150, 300, or 600 mg N-nitrosohepatamethyleneimine (NHMI)/kg, cells were harvested from tracheas, esophagi, and lungs (target tissues for NHMI) of inbred F344 rats and seeded into culture dishes. Normal cells from nonexposed organs produced no proliferative epithelial foci (EF). Of those tracheas sampled 3 weeks following exposure to 150 and 300 mg/kg 10 and 20%, respectively, contained one or more EF that could be subcultured. Of these tracheas harvested 3 weeks post exposure to 600 mg/kg or 20 weeks post exposure to 150-600 mg/kg, 80-100% contained EF that could be subcultured. Twenty weeks after 600 mg NHMI/kg, the incidence of tracheas harboring cell populations with neoplastic potential (agarose-positive EF) was 80%, whereas the tracheal tumor incidence determined at 24 months was only 29%. Epithelial focus-forming units with various abnormal in vitro growth potentials were also detected in esophagi and lungs of NHMI-exposed rats.

Epithelial lesions induced by N-nitrosoheptamethyleneimine in host and transplanted rat tracheas.

N-Nitrosoheptamethyleneimine (NHMI) was given by gastric intubation, at a dosage of 10 mg/kg body weight twice weekly for 5-20 weeks, to F344 rats bearing subcutaneous tracheal transplants. Groups of 5 rats were killed at 5, 10, 15, and 20 weeks, and 1 group was killed at week 33 of the experiment (13 weeks after the last NHMI dose). Sequential changes in host tracheas consisted of hyperplasia with complete loss of mucociliary epithelium, squamous metaplasia, intense mononuclear infiltration, reestablishment of mucocillary epithelium (during the course of NHMI administration) except for focal areas of hyperkeratotic squamous metaplasia or marked dystrophic changes, and finally, papillomas, polyps, and invasive squamous cell carcinomas. Lesions in tracheal transplants consisted mostly of atrophic and dystrophic changes, with only a few small foci of squamous metaplasia and no neoplastic changes.

Chronic inhalation of cigarette smoke by F344 rats.

Specific-pathogen-free female F344 rats were exposed by inhalation to what was considered a maximal tolerated dose of cigarette smoke. Total pulmonary deposition of smoke particulates from a single cigarette was 0.25 mg in young rats. Rats were exposed to smoke from 7 cigarettes/day for as long as 2.5 years, at which time 30% of the rats remained alive. Mortality of smoke-exposed animals was not different from that of untreated or sham-exposed controls. Hyperplastic and metaplastic areas in the epithelium of the nasal turbinates, larynges, and tracheae of exposed animals were observed at death. The lungs of exposed rats contained areas of focal alveolitis consisting of accumulated pigmented macrophages, epithelial hyperplasia, fibrosis, and disrupted alveolar structure. Smoke exposure did not change the total number of tumor-bearing animals relative to controls; however, exposed rats had significantly fewer tumors in the hypophyses, hematopoietic-lymphoid systems, uteri, and ovaries but an increased number of tumors in the respiratory tracts and dermes. Only 1 of 93 (1%) control rats had a tumor (an alveologenic carcinoma) in the respiratory tract as opposed to 7 of 80 (9%) exposed animals (nasal tumors: 1 adenocarcinoma and 1 squamous cell carcinoma; pulmonary tumors: 5 adenomas, 2 alveologenic carcinomas, and 1 squamous carcinoma).

Effect of carcinogen release rate on the incidence of preneoplastic and neoplastic lesions of the respiratory tract epithelium in rats.

Inbred F34 rat tracheal transplants were exposed to 7,12-dimethylbenz[a]anthracene (DMBA) delivered at different release rates for intraluminal pellets made of various matrices to study the effect of carcinogen dose rate on the induction of lesions in the epithelium. These matrices were beeswax, beeswax-stearyl alcohol, and beeswax-cholesterol. In addition, DMBA absorbed onto carbon particles was dispersed in beeswax-stearyl alcohol. The fastest release was obtained from beeswax pellets from which 99% of the carcinogen (198 micrograms) was released in 4 weeks, and the slowest release was from DMBA absorbed on carbon at a ratio of 1:9 from which only 56% (113 micrograms) was released in 16 weeks. Morphometry of histologic sections showed marked differences in the percentage of luminal surface covered by dysplastic-neoplastic epithelium (i.e., 7.5% in the tracheas exposed to the fastest releasing pellets and 46.3% in the tracheas exposed to the slowest releasing pellets). An inverse linear correlation was found between the cumulative amount of DMBA relased from the different pellet matrices of 2 weeks and the incidence of dysplastic plus neoplastic lesions of tracheal epithelium at 16 weeks. The results indicate that lower doses of carcinogen delivered slowly are more effective in producing dysplastic plus neoplastic lesions than hgher doses delivered rapidly.

Precursor lesions of bronchogenic carcinoma.

Clinical investigations and experimental studies concerned with preneoplastic and early neoplastic lesions in the respiratory tract are discussed. The occurrence of preinvasive states of carcinoma in the bronchus has been recognized for over 20 years. Histopathological and cytological studies suggest that it might become possible to identify even earlier preneoplastic precursor lesions provided the proper tissue or cell markers can be found. Experimental models that could be useful in establishing a better understanding of the evolution of the neoplastic diseases in the respiratory tract are now available.

In vitro transformation of primary rat tracheal epithelial cells by 5-azacytidine.

The ability of the hypomethylating agent 5-azacytidine to transform normal primary rat tracheal epithelial cells was determined. A single 24-h exposure to 5-azacytidine resulted in the transformation of rat tracheal epithelial cells at concentrations of the cytosine analogue ranging from 1 to 4 microM. Doses of 5-azacytidine that produced these enhanced growth transformants were nonmutagenic as measured by the induction of 6-thioguanine or ouabain resistance. In addition, the hypomethylating agent 5-azadeoxycytidine also transformed primary rat tracheal epithelial cells, whereas 6-azacytidine, a cytosine analogue which does not induce DNA hypomethylation, did not. Enhanced growth transformants resulting from 5-azacytidine exposure continued to progress in the absence of further treatment to give rise to variants with indefinite growth capacity which ultimately became neoplastic. These results indicate that hypomethylating agents can transform normal cells and suggest that changes in DNA methylation may occur during the initial stages of neoplastic transformation.

In vitro neoplastic progression of rat tracheal epithelial cells transformed by diverse carcinogens.

Preneoplastic transformants were isolated from primary rat tracheal epithelial cells after treatment with (a) mutagenic concentrations of the alkylating agent N-methyl-N-nitro-N'-nitrosoguanidine, (b) nonmutagenic concentrations of the DNA hypomethylating agent 5-azacytidine, or (c) after arising spontaneously. We have addressed the question of whether preneoplastic transformants induced by different carcinogens differ in their ability to progress to the immortal stage and to become neoplastic. Spontaneous transformants occurred with a very low frequency, and 5-azacytidine induced preneoplastic transformants half as efficiently as N-methyl-N-nitro-N'-nitrosoguanidine. However, no phenotypic differences could be detected between the 70 preneoplastic colonies isolated from the 3 groups; colony size, cell density, and clonogenicity were not statistically different. Clones from all 3 groups became immortal and further progressed to become neoplastic with similar frequencies. The level of expression of the oncogenes H-ras, K-ras, and raf was also similar in all 3 groups. These experiments indicated that there was no difference in the ability of spontaneous transformants or those induced by N-methyl-N-nitro-N'-nitrosoguanidine or 5-azacytidine to progress to become immortal or neoplastic. This suggests that whereas the nature of the carcinogen influenced the frequency of the initial transforming event, progression to the neoplastic stage was independent of the nature of the transforming insult.

Carcinogenicity of nickel subsulfide for respiratory tract mucosa.

The carcinogenicity of nickel subsulfide, Ni3S2, for respiratory tract epithelium was studied in heterotopic tracheal transplants with doses of 1 and 3 mg Ni3S2 per trachea. Chemical determinations indicated that Ni3S2 persisted in the tracheas for seven to nine months. Ni3S2 showed marked toxicity for mucociliary epithelium, resulting in widespread atrophy and focal epithelial necrosis during the first two months of exposure. The submucosa showed mononuclear infiltration and signs of fibroblastic and capillary proliferation. Tumor studies indicated that Ni3S2 can induce carcinomas in tracheal epithelium. The carcinoma incidence was 10% at 1 mg and approximately 1.5% at 3 mg. The higher dose produced a 67% incidence of fibro- and myosarcomas. The data suggest that, compared to some carcinogenic polycyclic hydrocarbons, Ni3S2 may not be a strong carcinogen for the epithelium of conducting airways. The data are discussed in light of other experimental studies and of epidemiological findings on respiratory tract cancers in nickel workers.

Increase in immunogenicity with concomitant loss of tumorigenicity of respiratory tract carcinomas during in vitro culture.

Cell lines were established in vitro from respiratory tract carcinomas induced in rats by carcinogenic, polycyclic hydrocarbons. Propagation of the carcinoma lines in vitro lead to a progressive decrease in tumorigenicity. Tumor transplantation studies in X-irradiated, immunosuppressed recipients and in immunologically reconstituted recipients suggested that the cells are rejected because of their immunogenicity, since a high incidence of tumors was observed in X-irradiated recipients but not in normal or X-irradiated, reconstituted recipients. When immunologically competent rats were immunized with cells from an in vitro tumor line, strong tumor transplantation resistance resulted. Similar immunization with the corresponding in vivo tumor line caused very little if any protection, and immunization with a non-cross-reacting sarcoma line grown in vitro did not produce immunological protection against carcinoma cell lines. A single in vivo passage of the in vitro-adapted tumor line in immunosuppressed recipients fully restored tumorigenicity. The increase in immunogenicity of carcinomas cultured in vitro appears to involve preexisting angigens indigenous to the carcinomas rather than new antigens acquired during tissue culture, such as antigens related to retroviruses, mycoplasmas, or heterologous serum.

Promotion-like enhancement of tracheal carcinogenesis in rats by 12-O-tetradecanoylphorbol-13-acetate.

Experiments were conducted to determine whether two-stage carcinogenesis could be observed in rat tracheal epithelium using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as promoter. Heterotopic tracheal transplants in Fischer 344 rats were first exposed to 188 microgram of DMBA delivered over a four-week period and subsequently to 100 microgram of TPA. The TPA was released from beeswax pellets at a rate of 1.1 microgram/day during the first two months and at a rate of 0.3 microgram/day for the subsequent two months. TPA alone caused marked inflammation and epithelial hyperplasia in tracheal grafts but no metaplastic or dysplastic changes. The tumor incidence in tracheas exposed to DMBA only was 20%; that in tracheas exposed to DMBA followed by TPA was 72%. TPA also accelerated the appearance of tumors. The mean tumor induction time in the group exposed to DMBA only was 91 weeks as compared to 75 weeks in the group exposed to DMBA and TPA. The data indicate that TPA enhances the tracheal tumor response in a manner similar to that of tumor promotion in mouse skin.

Demonstration of cross-reacting tumor rejection antigens in chemically induced respiratory tract carcinomas in rats.

Experiments were performed to determine whether chemically induced rat respiratory tract carcinomas, which possess considerable immunogenicity, contain cross-reacting antigens. In vivo studies showed that effective cross-protection can be induced with four of the five respiratory tract cancers studied, suggesting that these tumors have common tumor rejection antigens. In vitro cytotoxicity studies with sera from tumor-immune hosts showed cross-reactivity among three of the carcinomas tested.

Quantitative assessment of generalized epithelial changes in tracheal mucosa following exposure to 7,12-dimethylbenz(a)anthracene.

Sequential morphological changes occurring after brief carcinogen exposures of heterotopic tracheal transplants in rats were semiquantitatively studied. Tracheas were exposed to 7,12-dimethylbenz(a)anthracene for 1, 2, or 4 weeks, during which time means of 138, 152, and 160 microgram 7,12-dimethylbenz(a)anthracene, respectively, were delivered. The first two types of exposures resulted only in generalized epithelial changes; these included hyperplasia and early metaplasia, both of which regressed rapidly, and persistent atrophic alterations. No focal epithelial lesions or tumors developed. The third type of exposure (160 microgram 7,12-dimethylbenz(a)anthracene delivered in 4 weeks) resulted in the appearance of generalized mucosal changes with long-lasting, severe inhibition of mucus production. In addition, focal metaplastic lesions reappeared at 4 to 8 months after exposure, and invasive carcinomas developed after 1 year with an incidence of 9%. Overall carcinoma incidence, including carcinoma in situ, was 15%. The studies emphasize the importance of the duration of carcinogen exposure, and they demonstrate the emergence of focal lesions when effective carcinogenic exposures are being used. The possible significance of epithelial atrophy in the pathogenesis of cancer in this experimental model is discussed.

Demonstration of cellular and humoral immunity to transplantable carcinomas derived from the respiratory tract of rats.

Previous studies with respiratory tract tumors in mice have suggested that such tumors are not immunogenic or are only weakly so. To determine whether this is a general characteristic of neoplasias found in the airways of rodents, we investigated seven transplantable carcinomas in rats, six of which originated from tracheal epithelium and one of which orginated from the distal lung. These carcinomas were all of the squamous type and were induced by three different carcinogenic polycyclic hydrocarbons. All of the tumors were shown to be immunogenic, capable of mobilizing cellular and humoral immune responses in isogenic hosts upon immunization. This was demonstrated by induction of transplantation resistance, by Winn's neutralization test, and by the detection of antibodies in the sera of tumor-immune hosts by two independent methods (antibody-dependent cytotoxicity and antibody-binding test). The degree of immunogenicity varied among the tumor lines. The most metastatic tumor was clearly the least immunogenic. The relationship between carcinogenic insult and immunogenicity, as well as the possible nature of the tumor-associated antigens involved, is discussed.