The cytology of spontaneous neoplastic transformation in culture

Summary Thirteen cell lines derived from embryos of the mouse, rat and hamster were sampled at closely spaced intervals for cytology and spontaneous neoplastic transformation. Transformation, determined by growth of the cells as malignant neoplasms in compatible hosts, occurred in eight lines and appeared to be unrelated to time in culture, passage number, proliferative activity of the cells or type of serum used to supplement the culture medium. In an effort to establish cytologic criteria for malignancy of rodent fibroblasts, cultures were examined for 19 cytologic abnormalities. The percentage of cells or colonies showing each of the abnormal properties was recorded. Five of the 19 were found to correlate with neoplastic transformation in all cell lines. These were cytoplasmic basophilia, reduced cytoplasmic spreading on substrate, cording, high nuclear: cytoplasmic ratio and clumping. Each of these properties is amenable to quantitation or computerized image analysis. On the basis of the five cytologic criteria, the percentage of colonies diagnosed neoplastic, borderline and non-neoplastic was determined for each line. Although these percentages varied from one transplant generation to another, the percentage of neoplastic colonies tended to increase with time in all lines which became malignant, and the borderline colonies tended to decrease, suggesting a progression from borderline to neoplastic state.     

Growth of normal and neoplastic rat pleural mesothelial cells in the presence of conditioned medium from neoplastic mesothelial cells

Several transformed cells have been demonstrated to secrete growth factors. We studied the effect of conditioned medium from neoplastic rat pleural mesothelial cells on normal and neoplastic mesothelial cell growth. The results showed that the concentrated conditioned medium stimulated neoplastic mesothelial cell growth but inhibited reversibly normal mesothelial cell growth.     

Wistar大鼠的自发肿瘤病变及其发生率

目的了解Wistar大鼠的各种自发性肿瘤病变及其发生率。方法致癌实验中的对照组,采用4周龄SPF级Wistar大鼠,雌、雄性大鼠各60只,实验前观察1周,常规饲料喂饲104周后处死,进行组织病理学检查。结果报告了Wistar大鼠的各种自发性肿瘤病变及其发生率。雄性大鼠中发生肿瘤的动物占49.12%,发生良性肿瘤的动物占38.60%、发生恶性肿瘤的动物占17.54%;良性肿瘤主要有垂体腺瘤(19.30%)、睾丸间质细胞瘤(5.26%)和皮下纤维瘤(5.26%);恶性肿瘤主要有鳞状细胞癌(7.02%)和淋巴造血系统肿瘤(3.51%)。雌性大鼠中发生肿瘤的动物占60.34%;发生良性肿瘤的动物占50.00%、发生恶性肿瘤的动物占15.52%;良性肿瘤主要有乳腺纤维腺瘤(25.86%)和垂体腺瘤(24.14%);恶性肿瘤主要有腺癌(5.17%)和乳腺癌(3.45%)。结论本文报告的Wistar大鼠自发肿瘤及其发生率进一步丰富了现有SPF级Wistar大鼠自发性肿瘤的数据资料,可为有关技术人员提供一定的参考和借鉴。     

Serum-induced chromosome damage and neoplastic transformation of mouse cells in vitro

Summary In previous studies, mouse cells grown in medium supplemented with horse serum (HS) developed more chromosomal aberrations and underwent malignant transformation earlier than cells from the same pool grown with fetal bovine serum (FBS) supplement. In the present study cells derived from C3H_f/HeN mouse embryos were grown in medium NCTC-135 supplemented with various combinations of large- and small-molecule fractions of HS and FBS in an effort to determine the effective components. The results indicate that the large-molecule fraction of HS (mare or stallion) produces alterations in chromosome number and structure. HS is also shown to cause chromatid breaks and exchanges at or near the centromere in contrast to fluorescent-light-induced breaks which occur randomly along the chromatid. However, efforts to control completely chromosome stability and malignant transformation through the use of large-and small-molecule fractions of HS and FBS or combinations thereof were unsuccessful. In comnection with this study, diagnosis of malignant transformation in vitro was made by a direct sampling method based on cytologic criteria previously described and documented. With one exception, the diagnoses of 11 different cell lines were consistent with results of in vivo assays.     

Cellular immune competence of a Drosophila mutant with neoplastic hematopoietic organs

In the Tum^l mutant of Drosophila melanogaster, the larval hematopoietic organs undergo neoplastic changes and release into circulation large numbers of blood cells. The lamellocytes, and to a lesser extent the plasmatocytes from which they are derived, are the cells that encapsulate various endogenous tissues and form melanotic tumors. The mutation is temperature sensitive, with maximum gene expression manifested at 29°C. The ability of Tum^l larvae to encapsulate eggs of the wasp parasite Leptopilina heterotoma is dependent not only on temperature, with host larvae much more immune reactive at 29°C than at lower temperatures (15° or 21°C), but also on the interval of time following infection when temperature shift experiments are performed. When the shift of parasitized larvae from 21° to 29°C is delayed by 18 hr the hosts are not as immune reactive as those shifted immediately after infection. Since Tum^l larvae are potentially highly immune reactive at the time of infection (with sufficient numbers of lamellocytes in circulation to encapsulate parasites), the low degree of immune competence in hosts shifted to 29°C after 18 hr or maintained at lower temperatures suggests that the increased capacity of blood cells to react against foreign surfaces is dependent on the cells acquiring new or altered recognition and adherence properties at 29°C. The 18-hr delay may provide the parasite with an opportunity to interfere with the acquisition of these specific cellular alterations. Differential hemocyte counts from parasitized larvae show abnormally low lamellocyte counts in susceptible hosts, indicating that successfully developing parasites interfere with the differentiation of hemocytes.     

Relation of the slow growth phenotype to neoplastic transformation: Possible significance for human cancer

Summary Deletions are widely distributed over the genome in the most frequently occurring human cancers and are the most abundant genetic lesion found there. Deletions are highly correlated with the slow growth phenotype of mutated animal and human cells and result in chromosomal transposition when the retained ends are joined. Transpositions are only a minor source of mutation in rapidly multiplying bacteria but are a major cause of mutations in stationary bacteria. The NIH 3T3 line of mouse cells undergoes neoplastic transformation during prolonged incubation in a stationary state and expresses the slow growth phenotype on serial subculture at low density, suggesting a relation between transformation and chromosomal deletions. To further explore the relation between neoplastic transformation and the slow growth phenotype as a surrogate for deletions, two sublines of the NIH 3T3 cells with differing competence for transformation were serially subcultured in the stationary state at confluence and tested at each subculture for transformation and growth rate. Cell death in a fraction of the population and a heritable slowdown in proliferation of most of the survivors became increasingly pronounced with successive rounds of confluence. The reduction in growth rate was not proportional to the degree of transformation of the cultures, but all of the transformed cultures were slow growers at low density. All of the discrete colonies from cloning transformed cultures developed at a lower initial rate than control colonies under optimal conditions for growth, but they continued to grow at later stages, forming multilayered colonies under conditions that inhibited the further growth of the control colonies. The results suggest that prolonged incubation of NIH 3T3 cells in the stationary state results in growth-impairing deletions over a wide range of sites in the genome, but more restricted subsets of such lesions are responsible for neoplastic transformation. These findings provide dynamic, functional support in culture for the histopathological evidence that the quiescent state of cells associated with atrophy and fibrosis plays a significant role in the origin of some cancers in experimental animals and human beings.     

Alterations in the production of hemocytes due to a neoplastic mutation of Drosophila melanogaster

The hemocytes of a genetically induced, temperature-sensitive lethal mutation of Drosophila, Tum¹, were examined both quantitatively and qualitatively during the third larval instar. At the tumor-permissive temperature, 29°C, there was a fourfold increase in the concentration of circulating hemocytes in mutant larvae as compared to control. Additionally, the relative frequency of lamellocytes was 30 times greater in Tum¹ larvae than Basc in the early third instar. However, the severity of this abnormality gradually diminished as Tum¹ approached pupariation; though high frequencies of lamellocytes were always present. At the tumor-restrictive temperature (15°C) the concentration of circulating hemocytes was over twice that found at 29°C for Tum¹ larvae, and did not change during the course of third instar. However, in contrast to 29°C there was no abnormal increase in the frequency of lamellocytes at the tumor-restrictive temperature. Control larvae had equivalent concentrations of hemocytes at both temperatures. In one of two temperature shift experiments, Tum¹ larvae shifted from 15° to 29°C at the beginning of third instar expressed the abnormal hemocyte concentration and differentiation associated with larvae raised continuously at 29°C. In addition, Tum¹ larvae shifted from 29° to 15°C expressed reduced abnormalities of hemocyte differentiation, e.g., with fewer lamellocytes in circulation. The possibility of a temperature-sensitive period for the activation of the Tum¹ gene is discussed.     

Feline sarcoma virus induced in vitro progression from premalignant to neoplastic transformation of human diploid cells

Summary Human diploid cells morphologically transformed by feline sarcoma virus were serially propagated under selective cell culture conditions. When injected into nude mice prior to passage in soft agar (0.35%), morphologically transformed cells did not produce tumors. However, when propagated under selective cell culture conditions, transformed cells grew in soft agar and, when injected subcutaneously into the subcapsular region of the nμ/nμ mice, produced neoplastic nodules histopathologically interpreted as fibromas. Karyological examination of cell populations grown out from the tumors confirmed that the tumors were composed of human cells. Examination of electron micrographs of the excised tumor tissue revealed the presence of budding virus particles. Tumor cells isolated from nude mice and morphologicaly transformed cells both contained the feline concornativirus-associated cell membrane antigen. It was concluded that expression of feline oncornavirus-associated cell membrane antigen is associated with an early stage of feline rerovirus-induced carcinogenesis, namely focus formation. In addition, it was shown that FeLV-FeSV can induce morphological transformation in human cells in vitro and that there is a requirement for the cells to passage through soft agar before subsequent tumor formation (neoplastic transformation) can be demonstrated. This work was supported in part by NIH-NCI RO1-259007, NO1-CP-3571 and CPV08 103563, and Air Force F49620-77-C-110.     

Different induction of adaptive response to ionizing radiation in normal and neoplastic cells

Since the beneficial effects of low-dose radiation (0.01 Gy) are usually observed in normal cells, we investigated whether the adaptive response was induced by low-dose radiation in neoplastic cells of different origin as well as in normal cells. Cell lines used in this experiment were as follows: mouse lymphocytes (NL); L929 cells established from mouse connective tissue; primary mouse keratinocytes (PK); line 308 from mouse papilloma; X-ray sensitive lymphoma cells, L5178Y-S and EL-4 cells from mouse lymphoma. The adaptive response was determined by cell survival and apoptosis. The involvement of apoptosis in the adaptive response was examined by ELISA and TUNEL assay. Adaptive response was induced by pretreatment with low-dose radiation of 0.01 Gy in normal cells such as NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. In addition, the reduction of apoptosis by pretreatment with low-dose radiation was observed in NL, L929, and PK, but not in L5178Y-S, EL-4, and line 308 cells. These results suggested that the adaptive response could be induced by pretreatment with low-dose radiation and the phenomena were observed in normal cells, not in neoplastic cells. In addition, pretreatment with low-dose radiation reduced apoptosis, suggesting that an anti-apoptotic pathway may be involved in the adaptive response. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interaction of serum and cell spreading affects the growth of neoplastic and non-neoplastic fibroblasts

Both growth factor availability and cell-to-cell contact have been mechanisms used to explain cell growth regulation at high cell density. Recently Folkman and colleagues have shown that changes in cell shape, rather than cell-to-cell contact, can regulate the growth of fibroblasts. However, in those studies the relation between serum and shape regulation of growth was not studied, nor were neoplastic and non-neoplastic cells compared. In this report we have studied these aspects by varying cell spreading and serum concentration independently for 2 non-neoplastic and 3 neoplastic cell lines. Cell spreading (projected cell area) was controlled by decreasing the adhesiveness of tissue culture plastic plates with poly (hydroxyethyl methacrylate) [poly (HEMA)]. Cell growth was measured as the increase in cell number/day. We have found that more spreading increased net growth of both neoplastic and non-neoplastic cells, while less spreading (toward rounded configuration) depressed growth. There were also quantitative differences between neoplastic and non-neoplastic cells. Neoplastic cells continued to grow under conditions of cell rounding, which completely prevented the growth of their non-neoplastic counterparts. Some neoplastic cells also tended to show little or no increase in net cell number for serum concentrations above 10% as cells became more spread; in contrast, all non-neoplastic cells grew more with increasing concentrations of serum as they became well spread. Thus, in normal cells, it appears that the sensitivity of cells to humoral factors is governed by cell spreading. This interaction between serum and cell shape is less prominent in some neoplastic cells.     

The reduced extracellular calcium requirement for proliferation by neoplastic hepatocytes

Summary Cells from neonatal rat livers were unable to maintain DNA-synthetic activity in calcium-deficient medium, but neoplastic hepatocytes from Morris hepatomas 5123 tc and 7795 synthesized DNA and proliferated indefinitely in this calcium-deficient medium. The calcium content of fresh hepatoma tissue from which these cultures were derived was as much as 10 times greater than that of normal liver; but this difference could not account for the insensitivity of neoplastic cells to extracellular calcium because it disappeared during subsequent cultivation in vitro. NRCC No. 16597     

Collective biology of neoplastic disease in dicotyledonous plants

I discuss the two different responses from the angiosperms to the specific molecular mechanisms of the tumor-inducing agent contained in the bacteriumAgrobacterium tumefaciens. This is done in terms of the collective variables for expressing genetic response to a continuously varying supply of energy from metabolic pathways. We are led to the conjecture that the expression of the recessive oncogenes may not be restricted to humans (retinoblastoma and osteosarcoma), but may also occur in plants (crown gall), and be expressed through a heat-shock.     

Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture

The biology of animal cells is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three-dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three-dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial issues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.     

Transforming growth factors and control of neoplastic cell growth

Transforming growth factors (TGFs) are peptides that affect the growth and phenotype of cultured cells and bring about in nonmalignant fibroblastic cells phenotypic properties that resemble those of malignant cells. Two types of TGFs have been well characterized. One of these, TGF alpha, is related to epidermal growth factor (EGF) and binds to the EGF receptor, whereas the other, TGF beta, is not structurally or functionally related to TGF alpha or EGF and mediates its effects via distinct receptors. TGF beta is produced by a variety of normal and malignant cells. Depending upon the assay system employed, TGF beta has both growth-inhibitory and growth-stimulating properties. Many of the mitogenic effects of TGF beta are probably an indirect result of the activation of certain growth factor genes in the target cell. The ubiquitous nature of the TGF beta receptor and the production of TGF beta in a latent form by most cultured cells suggests that the differing cellular responses to TGF beta are regulated either by events involved in the activation of the factor or by postreceptor mechanisms. The combined effects of TGF beta with other growth factors or inhibitors evidently play a central role in the control of normal and malignant cellular growth as well as in cell differentiation and morphogenesis. Since transforming growth factor as a concept has partially proven misleading and insufficient, there is a need to find a new nomenclature for these regulators of cellular growth and differentiation.     

Arthropod-induced neoplastic formations on trees change photosynthetic pigment levels and oxidative enzyme activities

Abstract

In order to test the hypothesis that arthropod-induced neoplastic formations on trees affect biochemical characteristics of both the newly formed galls and host plant tissues, biochemical characteristics with a possible adaptive role were determined in nine gall-former–host tree combinations. Photosynthetic pigments, extractable protein content, and oxidative enzyme activities were determined in gall tissues, leaf tissues of galled leaves, and leaves on ungalled tree branches. Neoplastic tissues were characterized by a low content of photosynthetic pigments, decreased chlorophyll a/b ratio, lower extractable protein content, and decreased activities of peroxidase and polyphenol oxidase as compared with ungalled host leaf tissues. In galled leaves or in leaves adjacent to galls, increased level of peroxidase activity was found. In several gall-inducer–host plant combinations, galled host plant tissues contained increased activity of polyphenol oxidase as well. The presented data reflect long-term systemic effects of neoplastic formation on host tree physiology suggesting that gall inducers affect potential adaptive responses of host plants.     

Special symposium: In vitro plant recalcitrance loss of plant organogenic totipotency in the course of In vitro neoplastic progression

Summary The aptitude for organogenesis from normal hormone-dependent cultures very commonly decreases as the tissues are serially subcultured. The reasons for the loss of regenerative ability may vary under different circumstances: genetic variation in the cell population, epigenetic changes, disappearance of an organogenesis-promoting substance, etc. The same reasons may be evoked for the progressive and eventually irreversible loss of organogenic totipotency in the course of neoplastic progressions from hormone-independent tumors and hyperhydric teratomas to cancers. As in animal cells, plant cells at the end of a neoplastic progression have probably undergone several independent genetic accidents with cumulative effects. They indeed are characterized by atypical biochemical cycles from which they are apparently unable to escape. The metabolic changes are probably not the primary defects that cause cancer, rather they may allow the cells to survive. How these changes, namely an oxidative stress, affect organogenesis is not known. The literature focuses on somatic mutations and epigenetic changes that cause aberrant regulation of cell cycle genes and their machinery.     

Inhibition of neoplastic cell growth by quiescent cells is mediated by serum concentration and cAMP phosphodiesterase inhibitors

We have demonstrated that confluent monolayers of the mouse fibroblast cell line C3H/10T1/2 (10T1/2) have the ability to cause reversible growth inhibition of cocultured transformed cells. This was first demonstrated for de novo transformed cells and later extended to established cell lines of proven oncogenicity in vivo. This growth inhibition could be increased by growing the 10T1/2 cells to high density in increasing concentrations of serum or by elevating intracellular concentrations of cAMP using inhibitors of phosphodiesterase (PDE). These manipulations, which in cocultures of nontransformed and transformed cells caused complete inhibition of tumor cell growth, had no effect on growth rate or saturation density of either ceil type when cultured alone, demonstrating the cooperative nature of this phenomenon. This cooperation could not be produced by transfer of culture medium, demonstrating the requirement for intimate cell contact. Inhibition of the formation of transformed foci of cells in these mixed cultures was accompanied by a decrease in the incorporation of labeled thymidine into these cultures; the kinetics of this inhibition and recovery suggested a rapidly reversible effect on cell cycle transit times. The potent inhibitor of cAMP PDE, Ro 20-1724 induced dose dependent increases in intracellular cAMP in both nontransformed and in transformed cells. However, at a concentration of 10^?4 M Ro 20-1724, which inhibited tumor cell growth in mixed cultures, cAMP was elevated 30-fold in nontransformed versus only 3-fold in transformed cells. The inhibitory effects of PDE inhibitors on tumor growth have been extended to an in vivo model system, utilizing Lewis lung carcinoma cells growing as metastases in the lungs of C57B1 mice. In these mice, inoculated intravenously with a single cell suspension of Lewis lung cells, the formation of lung metastases was dramatically decreased by the twice daily administration of either isobutylmethylxanthine or Ro 20-1724; PDE inhibitors were shown to be active in vitro. The latter compound, which showed highest activity in vitro, was also substantially more potent in vivo as an inhibitor of lung tumor colony formation and doubled the life span of the tumor bearing animals. Cell cycle analysis of lung tumor colonies by the labeled mitosis method showed that both phosphodiesterase inhibitors caused a prolonged G₁ phase in the cell cycle but failed to influence other phases. Although detailed analysis of host tissues is not complete, prolonged treatment with these drugs caused no statistically significant weight loss or changes in counts of red or white blood cells indicating a selective growth inhibition of transformed cells at these doses. Studies to determine the mechanism of the cellular communication and the nature of the signal are in progress.     

DNA methylation as a key process in regulation of organogenic totipotency and plant neoplastic progression?

Summary Progressive loss of organogenic totipotency appears to be a common event in long-term plant tissue culture. This loss of totipotency, which has been proposed to be a typical trait of plant neoplastic progression, is compared to some mechanisms that occur during the establishment of animal differentiation-resistant cancer lines in vitro. Evidence is presented that alteration in DNA methylation patterns and expression of genes occur during long-term callus culture. An effect of the auxin, 2,4-dichlorophenoxyacetic acid, in the progressive methylation, is moreover suggested. Methylation of genes relevant to cell differentiation and progressive elimination of cells capable of differentiation is proposed as being responsible for this progressive loss of organogenic potential. Finally, the epigenetic alteration (DNA methylation) that occurs during prolonged periods of culture may induce other irreversible genetic alterations that ultimately make the loss of totipotency irreversible.     

Raman spectroscopy can differentiate malignant tumors from normal breast tissue and detect early neoplastic changes in a mouse model

Raman spectroscopy shows potential in differentiating tumors from normal tissue. We used Raman spectroscopy with near-infrared light excitation to study normal breast tissue and tumors from 11 mice injected with a cancer cell line. Spectra were collected from 17 tumors, 18 samples of adjacent breast tissue and lymph nodes, and 17 tissue samples from the contralateral breast and its adjacent lymph nodes. Discriminant function analysis was used for classification with principal component analysis scores as input data. Tissues were examined by light microscopy following formalin fixation and hematoxylin and eosin staining. Discriminant function analysis and histology agreed on the diagnosis of all contralateral normal, tumor, and mastitis samples, except one tumor which was found to be more similar to normal tissue. Normal tissue adjacent to each tumor was examined as a separate data group called tumor bed. Scattered morphologically suspicious atypical cells not definite for tumor were present in the tumor bed samples. Classification of tumor bed tissue showed that some tumor bed tissues are diagnostically different from normal, tumor, and mastitis tissue. This may reflect malignant molecular alterations prior to morphologic changes, as expected in preneoplastic processes. Raman spectroscopy not only distinguishes tumor from normal breast tissue, but also detects early neoplastic changes prior to definite morphologic alteration.