Selective killing of preneoplastic and neoplastic cells by methotrexate with leucovorin

Three sublines of NIH 3T3 cells had the properties of non-neoplastic, preneoplastic, and neoplastic cells, respectively. The closer the cells were to neoplastic behavior, characterized by continuing growth at high density, the slower they multiplied at lower density. Under the conditions of high population density and low calf serum concentration used in the assay for transformed focus formation, the transformed or neoplastic cells were much more sensitive to killing by methotrexate (MTX) than were non-neoplastic cells in the same culture. This differential sensitivity of neoplastic cells was far more pronounced in molecular, cellular, and developmental biology medium 402 (MCDB 402) than in DMEM. It is associated with the presence in MCDB 402 of folinic acid, known clinically as leucovorin, which is a reduced form of the folic acid present in DMEM. Although leucovorin had been shown to selectively spare normal bone marrow and intestine in animals from the killing effect of MTX on tumor cells, we demonstrate the preferential killing of neoplastic over non-neoplastic cells of the same derivation. Neither neoplastic nor non-neoplastic cells were killed once they had stopped multiplying at their respective saturation densities. The development of the light foci characteristic of the preneoplastic cells was less sensitive to MTX than the formation of the dense foci produced by the fully neoplastic cells. The system should serve as a valuable model to establish basic principles and optimal conditions for selective killing of neoplastic cells by chemotherapeutic drugs.

Differentially expressed protein Pdcd4 inhibits tumor promoter-induced neoplastic transformation

An mRNA differential display comparison of mouse JB6 promotion-sensitive (P+) and -resistant (P−) cells identified a novel gene product that inhibits neoplastic transformation. The JB6 P+ and P− cells are genetic variants that differ in their transformation response to tumor promoters; P+ cells form anchorage-independent colonies that are tumorigenic, and P− cells do not. A differentially displayed fragment, A7-1, was preferentially expressed in P− cells at levels ≥10-fold those in P+ cells, making its mRNA a candidate inhibitor of neoplastic transformation. An A7-1 cDNA was isolated that was identical to murine Pdcd4 gene cDNAs, also known as MA-3 or TIS, and analogous to human H731 and 197/15a. Until now, the function of the Pdcd4 protein has been unknown. Paralleling the mRNA levels, Pdcd4 protein levels were greater in P− than in P+ cells. Pdcd4 mRNA was also expressed at greater levels in the less progressed keratinocytes of another mouse skin neoplastic progression series. To test the hypothesis that Pdcd4 inhibits tumor promoter-induced transformation, stable cell lines expressing antisense Pdcd4 were generated from parental P− cells. The reduction of Pdcd4 proteins in antisense lines was accompanied by acquisition of a transformation-sensitive (P+) phenotype. The antisense-transfected cells were reverted to their initial P− phenotype by overexpression of a Pdcd4 sense fragment. These observations demonstrate that the Pdcd4 protein inhibits neoplastic transformation.

Hormone-regulatable neoplastic transformation induced by a Jun-estrogen receptor chimera

The v-jun oncogene encodes a nuclear DNA binding protein that functions as a transcription factor and is part of the activator protein 1 complex. Oncogenic transformation by v-jun is thought to be mediated by the aberrant expression of specific target genes. To identify such Jun-regulated genes and to explore the mechanisms by which Jun affects their expression, we have fused the full-length v-Jun and an amino-terminally truncated form of v-Jun to the hormone-binding domain of the human estrogen receptor. The two chimeric proteins function as ligand-inducible transactivators. Expression of the fusion proteins in chicken embryo fibroblasts causes estrogen-dependent transformation.

Coculturing diverse clonal populations prevents the early-stage neoplastic progression that occurs in the separate clones

Most human cancers are of monoclonal origin and display many genetic alterations. In an effort to determine whether clonal expansion itself could account for the large number of genetic alterations, we compared spontaneous transformation in cloned and uncloned populations of NIH 3T3 cells. We have reported that progressive transformation of these cells, which is driven by the stress of prolonged contact inhibition at confluence, occurs far more frequently in cultures of recent monoclonal origin than in their uncloned progenitors. In the present work we asked how coculturing six clones at early and late stages of progression would affect the dynamics of transformation in repeated rounds of confluence. When coculture started with clones in early stages of transformation, marked by light focus formation, there was a strong inhibition of the progression to the dense focus formation that occurred in separate cultures of the individual clones. In contrast, when coculture started after the individual clones had progressed to dense focus formation, there was selection of transformants from the clone producing the largest and densest foci. Mixing the cells of a single clone with a large excess of uncloned cells from a subline that was refractory to transformation markedly decreased the size of dense foci from clones in transit from light to dense focus formation, but had much less effect on foci from clones with an established capacity for dense focus formation. The major finding of protection against progression by coculturing clones in early stages of transformation suggests that the expansion of a rogue clone in vivo increasingly isolates many of its cells from genetically stabilizing interactions with surrounding clones. Such clonal isolation might account for the increase in mutation rates associated with the dysplasia in colorectal adenomas that signifies the transition between benign and malignant growth.

The cellular ecology of progressive neoplastic transformation: A clonal analysis

A comparison was made of the competence for neoplastic transformation in three different sublines of NIH 3T3 cells and multiple clonal derivatives of each. Over 90% of the neoplastic foci produced by an uncloned transformed (t-SA′) subline on a confluent background of nontransformed cells were of the dense, multilayered type, but about half of the t-SA′ clones produced only light foci in assays without background. This asymmetry apparently arose from the failure of the light focus formers to register on a background of nontransformed cells. Comparison was made of the capacity for confluence-mediated transformation between uncloned parental cultures and their clonal derivatives by using two nontransformed sublines, one of which was highly sensitive and the other relatively refractory to confluence-mediated transformation. Transformation was more frequent in the clones than in the uncloned parental cultures for both sublines. This was dramatically so in the refractory subline, where the uncloned culture showed no overt sign of transformation in serially repeated assays but increasing numbers of its clones exhibited progressive transformation. The reason for the greater susceptibility of the pure clones is apparently the suppression of transformation among the diverse membership that makes up the uncloned parental culture. Progressive selection toward increasing degrees of transformation in confluent cultures plays a major role in the development of dense focus formers, but direct induction by the constraint of confluence may contribute by heritably damaging cells. In view of our finding of increased susceptibility to transformation in clonal versus uncloned populations, expansion of some clones at the expense of others during the aging process would contribute to the marked increase of cancer with age.

PAGE-1, an X chromosome-linked GAGE-like gene that is expressed in normal and neoplastic prostate, testis, and uterus

We have used a combination of computerized database mining and experimental expression analyses to identify a gene that is preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus, and placenta, as well as in prostate cancer, testicular cancer, and uterine cancer. This gene is located on the human X chromosome, and it is homologous to a family of genes encoding GAGE-like proteins. GAGE proteins are expressed in a variety of tumors and in testis. We designate the novel gene PAGE-1 because the expression pattern in the Cancer Genome Anatomy Project libraries indicates that it is predominantly expressed in normal and neoplastic prostate. Further database analysis indicates the presence of other genes with high homology to PAGE-1, which were found in cDNA libraries derived from testis, pooled libraries (with testis), and in a germ cell tumor library. The expression of PAGE-1 in normal and malignant prostate, testicular, and uterine tissues makes it a possible target for the diagnosis and possibly for the vaccine-based therapy of neoplasms of prostate, testis, and uterus.

Cytologically normal cells from neoplastic cervical samples display extensive structural abnormalities on IR spectroscopy: Implications for tumor biology

Fourier-transform IR (FT-IR) spectra of pelleted exfoliated cervical cells from patients with cervical cancer or dysplasia differ from those from normal women. To study the origin of these spectral changes, we obtained the FT-IR spectra of individual cervical cells from normal, dysplastic, and malignant cervical samples. Ninety five percent of normal superficial and intermediate cells displayed two distinct spectral patterns designated A and B, and 5% displayed an intermediate pattern, suggesting extensive structural heterogeneity among these cells. Parabasal and endocervical cells showed pattern B spectra. The spectra of malignant, dysplastic, and other abnormal cells also were characterized. Analysis of FT-IR spectra of over 2,000 individual cells from 10 normal females, 7 females with dysplasia, and 5 females with squamous cell carcinoma revealed that the spectra of normal-appearing intermediate and superficial cells of the cervix from women with either dysplasia or cancer differed from those of normal women. Chemometric and classical spectroscopic analysis showed a continuum of changes paralleling the transition from normalcy to malignancy. These findings suggest that (i) the structural changes underlying the spectroscopic changes are involved in or are a product of cervical carcinogenesis and (ii) the neoplastic process may be more extensive than currently recognized with morphological criteria. This approach may be useful for the structural study of neoplasia and also may be of help in the diagnosis or classification of cervical disorders.

Plasticity of the neoplastic phenotype in vivo is regulated by epigenetic factors

Age of host and transplantation-site microenvironment influence the tumorigenic potential of neoplastically transformed liver epithelial cells. Tumorigenic BAG2-GN6TF rat liver epithelial cells consistently form tumors at ectopic sites, but differentially express tumorigenicity or hepatocytic differentiation in the liver depending on host age and route of cell transplantation into the liver. Direct inoculation into host livers concentrates tumor cells locally, resulting in undifferentiated tumors near the transplantation site in both young (3-month-old) and old (18-month-old) rats. Transplantation-site tumors regress within 1 month in the livers of young rats, but grow progressively in old rats. However, inoculation of cells into the spleen distributes transplanted cells individually throughout the liver, resulting in hepatocytic differentiation by tumor cells with concomitant suppression of their tumorigenicity in young rats. When transplanted into livers of old rats by splenic inoculation, or when young hepatic-transplant recipients are allowed to age, hepatocytic progeny of BAG2-GN6TF cells proliferate to form foci, suggesting that the liver microenvironment of old rats incompletely regulates the proliferation and differentiation of tumor cell-derived hepatocytes. Upon removal from the liver, BAG2-GN6TF-derived hepatocytes revert to an undifferentiated, aggressively tumorigenic phenotype. We posit that the spectrum between normal differentiation and malignant potential of these cells reflects the dynamic interaction of the specific transformation-related genotype of the cells and the characteristics of the tissue microenvironment at the transplantation site. Changes in the tissue milieu, such as those that accompany normal aging, may determine the ability of a genetically aberrant cell to produce a tumor.

Inhibition of neoplastic development in the liver by hepatocyte growth factor in a transgenic mouse model.

Overexpression of the c-myc oncogene is associated with a variety of both human and experimental tumors, and cooperation of other oncogenes and growth factors with the myc family are critical in the evolution of the malignant phenotype. The interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in a transgenic mouse model has been analyzed. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Furthermore, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice, whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine.

Irreversibility of cellular aging and neoplastic transformation: a clonal analysis.

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in a persistent impairment of proliferation when the cells are subcultured at low density and a greatly increased probability of neoplastic transformation in assays for transformation. These properties, along with the large accumulation of age pigment bodies in the confluent cells, are cardinal cellular characteristics of aging in organisms and validate the system as a model of cellular aging. Two cultures labeled alpha and beta were obtained after prolonged confluence; both were dominated by cells that were both slowed in growth at low population density and enhanced in growth capacity at high density, a marker of neoplastic transformation. An experiment was designed to study the reversibility of these age-related properties by serial subculture at low density of the two uncloned cultures and their progeny clones derived from assuredly single cells. Both uncloned cultures had many transformed cells and a reduced growth rate on subculture. Serial subculture resulted in a gradual increase in growth rates of both populations, but a reversal of transformation only in the alpha population. The clones originating from both populations varied in the degree of growth impairment and neoplastic transformation. None of the alpha clones increased in growth rate on low density passage nor did the transformed clones among them revert to normal growth behavior. The fastest growing beta clone was originally slower than the control clone, but caught up to it after four weekly subcultures. The other beta clones retained their reduced growth rates. Four of the five beta clones, including the fastest grower, were transformed, and none reverted on subculture. We conclude that the apparent reversal of impaired growth and transformation in the uncloned parental alpha population resulted from the selective growth at low density of fast growing nontransformed clones. The reversal of impaired growth in the uncloned parental beta population was also the result of selective growth of fast growing clones, but in this case they were highly transformed so no apparent reversal of transformation occurred. The clonal results indicate that neither the impaired growth nor the neoplastic transformation found in aging cells is reversible. We discuss the possible contribution of epigenetic and genetic processes to these irreversible changes.

Neoplastic development: paradoxical relation between impaired cell growth at low population density and excessive growth at high density.

The role of heritable, population-wide cell damage in neoplastic development was studied in the 28 L subline of NIH 3T3 cells. These cells differ from the 17(3c) subline used previously for such studies in their lower frequency of "spontaneous" transformation at high population density and their greater capacity to produce large, dense transformed foci. Three cultures of the 28 L subline of NIH 3T3 cells were held under the constraint of confluence for 5 wk (5 wk 1 degree assay) and then assayed twice in succession (2 degrees and 3 degrees assays) for transformed foci and saturation density. After the 2 degrees assay, the cells were also passaged at low density to determine their exponential growth rates and cloned to determine the size and morphological features of the colonies. Concurrent measurements were made in each case with control cells that had been kept only in frequent low-density passages and cells that had been kept at confluence for only 2 wk (2 wk 1 degree). Two of the three cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures produced light transformed foci, and the third produced dense foci. The light focus-forming cultures grew to twice the control saturation density in their 2 degrees assay and 6-8 times the control density in the 3 degrees assay; saturation densities for the dense focus formers were about 10 times the control values in both assays. All three of the cultures transferred from the 2 degrees assay of the 5 wk 1 degree cultures multiplied at lower rates than controls at low densities, but the dense focus formers multiplied faster than the light focus formers. The reduced rates of multiplication of the light focus formers persisted for > 50 generations of exponential multiplication at low densities. Isolated colonies formed from single cells of the light focus formers were of a lower population density than controls; colonies formed by the dense focus formers were slightly denser than the controls but occupied only half the area. A much higher proportion of the colonies from the 5 wk 1 degree cultures than the controls consisted of giant cells or mixtures of giant and normal-appearing cells. The results reinforce the previous conclusion that the early increases in saturation density and light focus formation are associated with, and perhaps caused by, heritable, population-wide damage to cells that is essentially epigenetic in nature. The more advanced transformation characterized by large increases in saturation density and dense focus formation could have originated from rare genetic changes, such as chromosome rearrangements, known to occur at an elevated frequency in cells destabilized by antecedent cellular damage.

Alterations in Notch signaling in neoplastic lesions of the human cervix.

The development of cancer is a cellular process that reflects and is partly driven by alterations in cell determination. Mutations in various molecules responsible for cell determination have been identified as being oncogenic, but little is known about the involvement of normal cell fate-determining mechanisms in the oncogenic process. The Notch pathway defines an evolutionarily conserved, general cell interaction mechanism that controls fundamental aspects of cell determination during vertebrate and invertebrate development. We have explored the involvement of the human Notch pathway in human cervical tissues, which define a cellular environment where cell fate changes take place and where neoplastic conditions have been well characterized. Our evidence suggests that Notch expression is associated with cell populations that are undergoing cell fate changes and that Notch activity can be used to monitor cell fate abnormalities in cervical as well as other epithelial neoplasias.

Heritable, population-wide damage to cells as the driving force of neoplastic transformation.

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in the death of some cells in a manner suggestive of apoptosis. Successive rounds of prolonged incubation at confluence of the surviving cells produce increasing neoplastic transformation in the form of increments in saturation density and transformed focus formation. Cells from the postconfluent cultures are given a recovery period of various lengths to remove the direct inhibitory effect of confluence before their growth properties are studied. It is found that with each round of confluence the exponential growth rate of the cells at low densities gets lower and the size of isolated colonies of the same cells shows a similar progressive reduction. The decreased growth rate of cells from the third round of confluence persists for > 60 generations of growth at low density. The proportion of colonies containing giant cells is much higher after a 2-day recovery from confluence than after a 7-day recovery. Retardation of growth at low density and increased saturation density appear to be two sides of the same coin: both occur in the entire population of cells and precede the formation of transformed foci. We propose that the slowdown in growth and the formation of giant cells result from heritable damage to the cells, which in turn drives their transformation. Similar results have been reported for the survivors of x-irradiation and of treatment with chemical carcinogens and are associated with the aging process in animals. We suggest that these changes result from free radical damage to membrane lipids with particular damage to lysosomes. Proteases and nucleases would then be released to progressively modify the growth behavior and genetic stability of the cells toward autonomous proliferation.

Detection and characterization of carcinoma cells in the blood

A highly sensitive assay combining immunomagnetic enrichment with multiparameter flow cytometric and immunocytochemical analysis has been developed to detect, enumerate, and characterize carcinoma cells in the blood. The assay can detect one epithelial cell or less in 1 ml of blood. Peripheral blood (10–20 ml) from 30 patients with carcinoma of the breast, from 3 patients with prostate cancer, and from 13 controls was examined by flow cytometry for the presence of circulating epithelial cells defined as nucleic acid+, CD45−, and cytokeratin+. Highly significant differences in the number of circulating epithelial cells were found between normal controls and patients with cancer including 17 with organ-confined disease. To determine whether the circulating epithelial cells in the cancer patients were neoplastic cells, cytospin preparations were made after immunomagnetic enrichment and were analyzed. Epithelial cells from patients with breast cancer generally stained with mAbs against cytokeratin and 3 of 5 for mucin-1. In contrast, no cells that stained for these antigens were observed in the blood from normal controls. The morphology of the stained cells was consistent with that of neoplastic cells. Of 8 patients with breast cancer followed for 1–10 months, there was a good correlation between changes in the level of tumor cells in the blood with both treatment with chemotherapy and clinical status. The present assay may be helpful in early detection, in monitoring disease, and in prognostication.

Regulation of transport pathways in tumor vessels: Role of tumor type and microenvironment

Novel anti-neoplastic agents such as gene targeting vectors and encapsulated carriers are quite large (approximately 100–300 nm in diameter). An understanding of the functional size and physiological regulation of transvascular pathways is necessary to optimize delivery of these agents. Here we analyze the functional limits of transvascular transport and its modulation by the microenvironment. One human and five murine tumors including mammary and colorectal carcinomas, hepatoma, glioma, and sarcoma were implanted in the dorsal skin-fold chamber or cranial window, and the pore cutoff size, a functional measure of transvascular gap size, was determined. The microenvironment was modulated: (i) spatially, by growing tumors in subcutaneous or cranial locations and (ii) temporally, by inducing vascular regression in hormone-dependent tumors. Tumors grown subcutaneously exhibited a characteristic pore cutoff size ranging from 200 nm to 1.2 μm. This pore cutoff size was reduced in tumors grown in the cranium or in regressing tumors after hormone withdrawal. Vessels induced in basic fibroblast growth factor-containing gels had a pore cutoff size of 200 nm. Albumin permeability was independent of pore cutoff size. These results have three major implications for the delivery of therapeutic agents: (i) delivery may be less efficient in cranial tumors than in subcutaneous tumors, (ii) delivery may be reduced during tumor regression induced by hormonal ablation, and (iii) permeability to a molecule is independent of pore cutoff size as long as the diameter of the molecule is much less than the pore diameter.