Maturation arrest of stem cell differentiation is a common pathway for the cellular origin of teratocarcinomas and epithelial cancers.

Analysis of the cellular origin of carcinomas of different organs indicates that there is in each instance, a determined stem cell required for tissue renewal that is the cell of origin for carcinomas. The normal tissue-determined stem cells are the result of differentiation in the embryo and are little changed, if at all, from the embryonic cells. Malignant stem cells are derived from these normal stem cells of adult tissues. The resultant tumors are caricatures of the normal process of tissue renewal with many stem cells and imperfect differentiation (14). This imparts an undifferentiated appearance to the tumors, not a dedifferentiated one. Study of the regulation of normal stem cells in the embryo should lead to rational therapies for malignant ones, and conversely, study of secretions and regulation of malignant stem cells will provide insights into normal regulation. The cancer-derived differentiated cells are benign (12, 74) if not normal (39, 53) leading to the conclusion that attempts to direct normal differentiation of malignant stem cells might serve as an alternative to cytotoxic therapy. Attempts to develop such therapies are currently underway (208). The degree of differentiation of a carcinoma depends on the proportion of undifferentiated tumor stem cells, the stage of maturation arrest of the majority of cells in the tumor, and on the ability of some cells to escape arrest and to differentiate (Fig. 1). These concepts of the stem cell contribution to tumors originated largely from studies of teratocarcinoma (209) and were not widely accepted because many considered the lessons learned were unique to teratocarcinomas and would not apply to other tissues. On the basis of the concepts covered in this review, it is clear that teratocarcinomas are unique only in the potential of their stem cells. Other stem cells have more limited potential. The balance of expression of the differentiated histiotype of the tumor cell lineage and the undifferentiated phenotype of the tumor stem cells determine the morphology of the tumor. Normal tissue renewal of epithelial organs is also from stem cells or their differentiating progeny. The cellular events during liver development and regeneration and the changes that precede the development of liver cancer during hepatocarcinogenesis are similar to the cellular response in pancreas, prostate, breast, lung, and gut. In liver, as in the leukopoietic system, the primitive tissue-specific stem cell is not primarily involved in renewal because that would be too slow a process; individuals would die before generation of sufficient replacement cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrogen peroxide as a mediator of programmed cell death in the blastocyst.

Previous work identified in blastocele fluid a soluble activity which killed embryonal carcinoma cells with trophectodermal potential but not those with embryonic potential [35]. From use of a malignant caricature of the late blastocyst, this toxic activity was postulated to be H2O2 [8]. The purpose of this paper was to determine if blastocele fluid also contained amounts of H2O2 capable of mediating the preferential killing of malignant pretrophectodermal cells (ECa 247). We not only observed that blastocele fluid is not toxic for these cells in the presence of catalase, but that malignant cells with embryonic potential (P19) that normally survive exposure to blastocele fluid become sensitive to it if their intracellular glutathione levels are lowered. Thus, it is concluded that the blastocyst contains amounts of H2O2 toxic to malignant pretrophectodermal cells and that glutathione-dependent mechanisms protect malignant inner cell mass cells with embryonic potential. Apparently, H2O2 production and glutathione-dependent protection mechanisms are developmentally regulated in the inner cell mass. These results are discussed with regards to apoptosis and the regulation of tissue mass.

Serum amine oxidase activity contributes to crisis in mouse embryo cell lines.

This paper reports the results of experiments to test the hypothesis that crisis of spontaneous transformation is caused by the hydrogen peroxide and/or aldehydes generated from endogenous polyamines by serum amine oxidase [amine: oxygen oxidoreductase (deaminating), EC 1.4.3.6]. After 4-5 weeks of culture, crisis occurred in 16 of 29 cell lines derived from limb buds of embryos from SJL/J, C3H, and CD-1 mice. In contrast, after the same time in culture but in medium supplemented with aminoguanidine, which inhibits serum amine oxidase, crisis occurred in only 1 of 41 cell lines. Protection against crisis was maximal in cell lines of SJL/J embryos, in which the incidence of crisis fell from 7 of 9 in untreated controls of 0 to 12 in the presence of 2 mM aminoguanidine. 2-Mercaptoethanol at 150-300 microM, which protects cells from serum amine oxidase-dependent polyamine toxicity, also protected the cell lines against crisis. These protected cell lines retained proliferative potential, diploid DNA content, and the mixture of cell types found in the primary cultures. These results indicate that cytotoxic catabolites generated by serum amine oxidase caused at least a large portion, but perhaps not all, of the cellular damage that leads to crisis in mouse embryo cell lines.

Neoplastic embryoid bodies of embryonal carcinoma C44 as a source of blastocele-like fluid.

There is a cytotoxic activity in blastocele fluid that kills embryonal carcinoma cells with trophectodermal potential but spares those with embryonic potential. This activity is present when programmed cell death occurs in the inner cell mass (ICM), and the ICM loses its trophectodermal potential. Because of the paucity of blastocele fluid, cystic embryoid bodies of embryonal carcinoma C44 were examined ultrastructurally and in tissue culture to determine if they corresponded to late blastocysts and if their fluid corresponded to blastocele fluid. No trophectoderm was demonstrated in the embryoid bodies, but embryonal carcinoma and endoderm were present, leading to the conclusion that the embryonal carcinoma corresponded to late ICM that had expressed endodermal potential. As a result the cyst fluid might have contained the toxic activity of blastocele fluid. The cyst fluid of C44 embryoid bodies did contain a soluble, low-molecular-weight, cytotoxic activity that preferentially killed embryonal carcinoma cells with trophectodermal potential while sparing those with embryonic potential. Enough of this fluid was available to determine the chemical nature of this toxic activity.

Evidence linking programmed cell death in the blastocyst to polyamine oxidation.

Programmed cell death occurs in the inner cell mass during blastulation concomitant with the loss of its trophectodermal potential, and blastocele fluid kills malignant inner cell mass cells with trophectodermal potential (ECa 247) but spares those with embryonic potential (P19). A previous study had shown that blastocele-like fluid from embryoid bodies of the teratocarcinoma C44 contains a low-molecular-weight cytotoxin that exhibits the same target-cell selectivity as normal blastocele fluid. The current paper shows that the preferential killing of cells with trophectodermal potential is caused by hydrogen peroxide generated during the oxidation of polyamines in the cyst fluid by amine oxidases. The greater resistance of cells with embryonic potential to hydrogen peroxide is due to glutathione-dependent mechanisms. These data lead to the conclusion that an amine oxidase in the blastocyst oxidizes polyamines in blastocele fluid, generating hydrogen peroxide which causes programmed cell death of normal and malignant cells with trophectodermal potential.

Amine oxidases, programmed cell death, and tissue renewal.

Embryonal carcinoma cells, with embryonic (ECaE) or trophectodermal (ECaT) potential, have been used in a colony assay to determine regulatory mechanisms in the blastocyst. The mechanism that regulates ECaE and results in chimera formation is dependent upon a soluble factor in blastocoele fluid and contact with trophectoderm. Two mechanisms contribute to the regulation of ECaT: one involves a factor in blastocoele fluid and the other contact with either trophectoderm or inner cell mass which results in differentiation of the cells into trophectoderm, and the other involves the killing of at least 40% of the cells by blastocoele fluid alone. This cytotoxic activity probably causes the programmed cell death that occurs in the inner cell mass during blastulation as it loses the potential to differentiate into trophectoderm. A toxic activity similar to that of normal blastocysts has been obtained from embryoid bodies. This activity is caused by amine oxidase-dependent catabolism of polyamines, and it is postulated that programmed cell death in the embryo and chalone activity in the adult may have similar mechanisms.

Polyamine oxidation, programmed cell death, and regulation of melanoma in the murine embryonic limb.

The murine embryonic limb at day 14 of gestation suppresses tumor formation by melanoma cells. Conditioned media of embryonic limbs have been found cytotoxic for B16 melanoma cells. The cytotoxicity is due to the catabolism of polyamines in the limb bud extracts by an amine oxidase in the serum supplement of the culture medium. However, a polyamine oxidase activity, similar to that in adult rat liver, is also detectable in homogenates of embryonic limbs. Thus, the embryonic limb contains the necessary components to produce polyamine-derived cytotoxic metabolites, which are present at the time programmed cell death occurs. This leads to the hypothesis that injected melanoma cells are killed incidentally by the mechanism that mediates programmed cell death.

Mechanism of programmed cell death in the blastocyst.

The malignant growth potential of embryonal carcinoma cells may be controlled by environmental factors. For example, embryonal carcinoma cells placed into normal blastocysts may not exhibit the continued growth expected of malignant cells but rather may lose all aspects of the malignant phenotype and become apparently normal embryonic cells. Loss of the malignant phenotype of embryonal carcinoma cells occurs early in these injected blastocysts and has been used as the basis of assays to study the mechanisms of regulation of embryonal carcinoma by the blastocyst. In this regard, P19, an embryonal carcinoma that makes midgestation chimeras, was regulated by blastocele fluid plus contact with trophectoderm but not by blastocele fluid plus contact with inner cell mass (ICM). In contrast, ECa 247, which makes trophectoderm, was regulated by exposure to blastocele fluid plus contact with trophectoderm or ICM. During the course of these experiments, dead embryonal carcinoma and ICM cells were observed, and blastocele fluid was then shown to kill ECa 247 and normal ICM cells of early blastocysts with trophectodermal potential. P19 cells and ICM cells with potential to make the embryo were not killed by blastocele fluid. Programmed cell death occurs in the ICM of the blastocyst during the transition from early (when ICM has the potential to make trophectoderm) to late (when the ICM lacks the potential to make trophectoderm). It is postulated that this programmed cell death is designed to eliminate redundant ICM cells with trophectodermal potential, and its mechanism of action is mediated by epigenetic factors in blastocele fluid.

Differentiation of ICM cells into trophectoderm.

It has been established previously that when inserted in the blastocyst E Ca 247 preferentially differentiates into trophectoderm in vitro. If the concept that tumors are caricatures of the process of tissue renewal is correct, then some cells from the inner cell mass (ICM), the normal counterpart of embryonal carcinoma, should be able to differentiate into trophectoderm. This has been a controversial issue. Four experiments are now reported that support the idea that ICM can differentiate into trophectoderm: 1) ICM from early blastocysts after classical immunosurgery made blastocysts in vitro; 2) ICM obtained from early blastocysts by immunosurgery using antigens other than histocompatibility ones made blastocysts in vitro; 3) ICM from early blastocysts, in which the trophectodermal cells had been labeled, contained no labeled cells following immunosurgery; and 4) In reconstruction experiments, polar and mural trophectodermal cells attached to ICM from late blastocysts failed to multiply and make blastocysts when cultured. It is concluded that like the embryonal carcinoma some ICM cells of early blastocysts have the potential to make trophectoderm. This fact is consistent with the concept that tumors are caricatures of the process of tissue renewal; and establishes E Ca 247 as a good model for study of trophectodermal differentiation.

Tumors as caricatures of the process of tissue renewal: prospects for therapy by directing differentiation.

A concept of neoplasms, based upon developmental and oncological principles, states that carcinomas are caricatures of tissue renewal, in that they are composed of a mixture of malignant stem cells, which have a marked capacity for proliferation and a limited capacity for differentiation under normal homeostatic conditions, and of the differentiated, possibly benign, progeny of these malignant cells. The concept brings order to the facts about carcinoma, has predictive value for embryogenesis, and indicates possibilities for differentiation therapy. One such possibility assumes on the basis of experimentation in vitro that malignant stem cells can be induced to differentiate into postmitotic cells by application of chemicals. Another suggests study of naturally occurring substances which regulate cell proliferation and differentiation in adult tissues. The other possibility, based upon experiments in vivo and in vitro, indicates that embryonic fields are capable of converting their closely corresponding malignant lineages into apparently normal lineages responsive to homeostatic control. Induced differentiation of embryonal carcinoma has been achieved in vivo with improvement in longevity of the host and in some cases with apparent cure. However, ultimate success of treatment based upon turning malignant cells into benign cells will depend upon the nature of the benign cells. Will they remain benign?

The fate of embryonal-carcinoma cells in mouse blastocysts.

Double-labeled embryonal-carcinoma (ECa) cells were injected into blastocysts or incorporated into blastocysts by aggregation, and their fate after various periods of time in culture was investigated. ECa-247 cells labeled with fluorescent microscopheres were easily identified in whole blastocysts. These blastocysts were embedded in plastic, serially sectioned, and prepared for autoradiography. The 3H-thymidine label on the embryonal-carcinoma cells allowed precise localization of the cancer-derived cells. ECa-247 cells preferentially localized in the mural trophectoderm, with a few being seen in primitive endoderm and, even more rarely, in the inner cell mass. Selected autoradiograms were re-embedded and thin sectioned for transmission electron microscopy. The cancer-derived cells were found to have differentiated in accordance with their localization.

Regulation of melanoma by the embryonic skin.

This report focuses on the regulation of murine melanoma by the embryonic skin. A surgical technique was developed to allow injection of B16 melanoma cells into the embryo in utero. A significant decrease in incidence of tumors was noted, which correlated with the time of arrival of normally migrating premelanocytes into the skin. Media were conditioned from skin explanted at the time premelanocytes arrive in it; these media inhibited the growth of melanoma cells in vitro. Under optimal conditions the growth of melanoma cells ceased; the cells had altered morphology and failed to proliferate when placed in fresh growth media.

The neurula stage mouse embryo in control of neuroblastoma.

The purpose of this study was to determine whether the neurula stage mouse embryo can regulate tumor formation of C-1300-3 neuroblastoma cells. Five neuroblastoma cells were injected into the second somite of neurula stage embryos, and their ability to form tumors was tested, 24 hr later, by transplanting the portion of the embryo containing the cancer cells into the testes of adult mice. Only one-third the number of tumors was obtained in comparison with controls in which (i) five neuroblastoma cells were injected into blocks of liver tissue that were then transplanted into the testes of adult animals or (ii) five C-1300-3 neuroblastoma cells were injected directly into the testes. When five C-1300-3 cells were injected into somites, which had been dissected from embryos, and the injected somites were placed in animals, significantly fewer tumors were obtained in relationship with controls. Although it is not known whether the neuroblastoma cells are induced to differentiate or are killed by the embryonic tissue, the effect appeared to be specific because the tumor-forming ability of L1210 leukemia, B-16 melanoma, embryonal carcinoma 247, and a parietal yolk sac carcinoma was unaffected by somites.

Trophectoderm in control of murine embryonal carcinoma.

It has been shown previously that the intact blastocyst of the mouse can regulate tumor formation and colony formation of murine embryonal carcinoma. This effect is consistent with the close histogenetic correspondence between embryonal carcinoma and the inner cell mass of the blastocyst. The ability of inner cell mass, blastocele fluid, and inner and outer surfaces of trophectoderm to abrogate colony formation of a variety of malignant tumors has now been tested. Direct contact of the embryonal carcinoma cells with the blastocele surface of trophectoderm proved to be necessary for abrogation of colony formation of embryonal carcinoma. This effect was not seen with any of the other tumors tested. Some tumors, which lack a normal cellular counterpart in the blastocyst, grew poorly in the blastocele unless a fistula was made in the wall of the blastocyst. Colony formation of the embryonal carcinoma was regulated in blastocysts with fistulas, but the other tumors were not regulated under these conditions. It is concluded that colony formation of embryonal carcinoma cells is regulated by direct contact with the trophectoderm of its corresponding embryonic field in an unknown but specific manner.