Contrasting roles of p57(KIP2) and p21(WAF1/CIP1/SDI1) in transplanted human and bovine adrenocortical cells.

Cell transplantation provides a way to compare the regulation of cell proliferation in the same cell type in cell culture and in a vascularized tissue structure in a host animal. The cyclin-dependent kinase inhibitors p57(KIP2), p21(WAF1/CIP1/SDI1) and p27(KIP1) have been extensively studied in cell culture but their role in growth control in tissues is less well understood. In the present experiments we compared the behavior of cell cycle inhibitors in human and bovine adrenocortical cells in culture and following cell transplantation in scid mice. p57 was expressed in the majority of cells in the intact human adrenal cortex. However, double immunofluorescence showed that cells that are in the cell cycle are p57(-) adrenocortical cells, p57 and p27 levels were not affected by inhibition of growth at high cell density, whereas p21 was higher in dividing than growth-inhibited cells. However, p21 was also high in senescent adrenocortical cells. After transplantation of human adrenocortical cells in scid mice, p57 and p27 were observed in most cells in the transplant tissue. Over time the number of p21(+) cells decreased greatly in human adrenocortical cells, but not in bovine adrenocortical cells. This difference correlated with lower levels of cell division (assessed by Ki-67 or incorporation of bromodeoxyuridine) in the human cells in transplant tissues in comparison to bovine cells. The differences between human and bovine cells were observed both when cells were transplanted beneath the kidney capsule and when cells were injected subcutaneously in collagen gel. We conclude that the behavior of p57, but not p21, is consistent with a role as a physiological mediator of proliferative quiescence in the adrenal cortex. The high level of p21 in dividing adrenocortical cells in culture, and in bovine adrenocortical cells in transplant tissues, may be a response to conflicting positive and negative growth influences. Cells may enter the cell cycle under the influence of a strong positive mitogenic signal, but coexisting negative growth stimuli trigger a p21-dependent block to further progression through the cell cycle. This model suggests that bovine adrenocortical cells respond to positive growth stimuli in transplant tissues but human cells lack this response.

Prevalence and distinctive biologic features of flat colorectal adenomas in a North American population.

BACKGROUND & AIMS: To assess the prevalence of flat and depressed (F&D) colorectal adenomas in the United States, we performed a prospective study of 211 American patients. METHODS: Dye-assisted colonoscopy was performed in the presence of both an American and a Japanese investigator. RESULTS: F&D lesions were found in 22.7% of patients, and these were more likely to be adenomatous than polypoid lesions (82% vs. 67%; P = 0.03) and contained more invasive cancer (4.5% vs. 0%; P = 0.04), which also appeared to be at a disproportionately advanced stage. The average size of all F&D advanced lesions (high-grade dysplasia and cancer) was significantly smaller than comparable polypoid lesions (10.75 +/- 2.7 mm vs. 20 +/- 2.9 mm; P < 0.05). F&D adenomas showed significantly stronger fragile histidine triad (FHIT) expression and lower p53 reactivity than similarly sized polypoid adenomas, whereas proliferative and apoptotic indices were similar in both groups. CONCLUSIONS: We conclude that there is a significant prevalence of colonic F&D colorectal adenomas in this country and that these lesions have significantly different biologic features than polypoid lesions. The clinical and epidemiologic implications of these findings for American patients need to be addressed in further studies.

CD13-positive anaplastic large cell lymphoma of T-cell origin--a diagnostic and histogenetic problem.

The expression of myelomonocytic-associated antigens in anaplastic large cell lymphomas (ALCLs), particularly those presenting in extranodal sites, can make their distinction from extramedullary myeloid cell tumors (EMCTs) or histiocytic tumors problematic. Yet, this distinction is clinically significant because of its therapeutic and prognostic implications. Herein, we describe a case of extranodal anaplastic lymphoma kinase-positive CD30-positive ALCL of T-cell origin in a 12-year-old boy, which was initially called an EMCT because of the expression of CD13 and HLA-DR detected by flow cytometry and the absence of other T-cell-related surface markers. However, the detection of cytoplasmic CD3 by flow cytometry prompted further studies. The tumor was composed of large cells with abundant slightly eosinophilic vacuolated cytoplasm and ovoid or reniform nuclei with a few small nucleoli. Using immunohistochemistry, the tumor was positive for CD45, CD30, CD45RO, and CD43 with a strong cytoplasmic and nuclear anaplastic lymphoma kinase stain. The tumor cells showed a T-cell clonal genotype. Electron microscopy revealed no ultrastructural features of myelomonocytic or histiocytic origin. The patient responded well to the chemotherapy and was in complete remission for 10 months at the time of submission of this manuscript. Review of the literature showed inconsistencies regarding the diagnosis, nomenclature, and, therefore, treatment and prognosis of these tumors. In addition, the CD13 expression in ALCL raises some histogenetic questions and may indicate origin from a pluripotent stem cell, misprogramming during malignant transformation, or a microenvironmental effect on lymphoid cell expression of surface antigens. Therefore, ALCL should be considered in the differential diagnosis of EMCTs or histiocytic tumors, particularly when surface marker lineage assignment is ambiguous.

Subcutaneous transplantation of bovine and human adrenocortical cells in collagen gel in scid mice.

Adrenocortical cells of bovine origin and of adult and fetal human origin were transplanted subcutaneously (s.c.) in scid mice after being embedded in collagen gel. In this site the cells survived, became vascularized by invasion of host endothelial cells, and secreted steroids into the circulation. The animals' own adrenal glands were removed at the time of cell transplantation. Steroids secreted by the transplants replaced the essential functions of the animals' own adrenal glands. Adrenalectomized animals without transplanted cells died after several days, but most animals with transplanted bovine or adult human adrenocortical cells survived; fewer animals survived with transplanted fetal human adrenocortical cells. The histology of the tissues formed from transplanted cells resembled that of the normal adrenal cortex. A few proliferating cells were observed in tissue from bovine or adult human cells; there was a greater percentage of dividing cells in tissue derived from fetal cells. Subcutaneous transplantation of bovine or human primary adrenocortical cells in collagen provides a model for the study of the physiology, cell biology, and molecular biology of adrenocortical cells in a three-dimensional vascularized tissue structure in a host animal.

Cell transplantation: a tool to study adrenocortical cell biology, physiology, and senescence.

We have established a mouse model for the growth and function of bovine and human adrenocortical cells in immunodeficient animals. We used the technique of cell transplantation, in which dispersed cells are introduced into an appropriate host in vivo to form a functional tissue. The ability to regenerate vascularized tissue, of normal histology and ultrastructure, is an inherent property of transplanted adrenocortical cells. Steroids secreted by the transplants replace the essential functions of the animals' own adrenal glands. Successful methods of transplantation described here have in common that the adrenocortical cells are permitted to aggregate in a space or matrix that provides adequate extracellular fluid and appropriate nutrients and oxygen. The present experiments show the potential of cell transplantation as a tool for the investigation of adrenocortical cell biology, molecular biology and physiology. The complete potential of the system will become apparent as new uses of the technique are devised, particularly with respect to human adrenocortical cells and to genetically modified cells.

Reconstituted human normal breast in nude mice using collagen gel or Matrigel.

Human breast epithelial cells dissociated from reduction mammoplasty specimens were embedded in two commonly used extracellular matrices, type I collagen gel or Matrigel, and subsequently transplanted subcutaneously into athymic nude mice. Histological sections from both types of recovered gels showed epithelial structures arranged as short tubules with some branching as well as preservation of epithelial cell polarity. Proliferation was studied in vivo by 5-bromo-2'-deoxy-uridine labeling followed by immunostaining of sections from recovered gels. Human breast epithelia embedded in collagen gel or Matrigel had similar proliferative activity. Cholera toxin, 17 beta-estradiol, and epidermal growth factor, when tested singly, were growth promoting, and in combination 17,beta-estradiol and cholera toxin had an additive effect but 17,beta-estradiol and epidermal growth factor were not additive. Our model system provides a means to study the endocrine control of normal human breast development.

In vivo growth stimulation of collagen gel embedded normal human and mouse primary mammary epithelial cells.

A new system for studying growth of normal human mammary epithelial cells in an in vivo environment using athymic nude mice is described. Human mammary epithelial cells dissociated from reduction mammoplasty specimens were embedded within collagen gels and subsequently transplanted subcutaneously into nude mice. Histological sections of recovered collagen gels showed epithelial cells arranged as short tubules with some branching. Proliferation of mammary epithelial cells was quantitated in vivo by 3 days' continuous infusion with 5 bromo-2'-deoxy-uridine followed by immunostaining of sections from recovered gels. Ovarian steroids administered to the host animals, resulting in blood serum levels normally found in the human female, had little or no effect on the proliferation of human mammary epithelial cells. Collagen gel embedded mouse mammary epithelial cells, mouse mammary explants, and host mammary glands all responded similarly to ovarian steroids, suggesting that the unresponsiveness of the human mammary epithelial cells under these conditions was not due to dissociation per se. However, an increased dose of 17 beta-estradiol or a growth factor combination containing epidermal growth factor, cholera toxin, and cortisol significantly stimulated the proliferation of human outgrowths. The growth factor response was dependent on the location of the cells, with the greatest response seen in the part of the gel proximal to the osmotic pump delivering the growth factors and the effect gradually waning in area more distal to the pump. The effect was especially striking since the mitotic figures could be easily identified and the labeling index was as high as 75%. The host mouse mammary gland also responded to growth factors, resulting in ductal hyperplasia. The proliferative and morphogenetic effects of various agents on normal human mammary epithelial cells embedded in collagen gel can be studied in vivo in nude mice.

Human breast cancers respond to growth factors in vivo but not in vitro.

Growth response of human breast cancer cells to epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) was tested both in culture and in vivo in nude mice. Human breast cancer cells were obtained from palpable tumors resulting from xenografted primary breast cancers in nude mice. In collagen gel culture, the breast cancer cells grew autonomously as expanding spherical masses of loosely adherent cells in the basal medium and the supplementation of growth factors had no additional stimulatory effect. To determine whether this in vitro response is reflected in vivo, the collagen gel embedded human breast cancer cells were transplanted into athymic nude mice and the growth response to EGF was studied in vivo. In contrast to the situation in vitro, exogenous EGF was growth promoting in vivo. Our results demonstrate the importance of the combined in vitro-in vivo approach in studying physiologically relevant growth regulation. In addition, the use of collagen gel embedded human breast cancer cells for transplantation studies may more closely model the clinical situation in view of the close histopathological resemblance of the recovered gels to the surgical breast specimens.