A synthetic antagonist to laminin inhibits the formation of osteolytic metastases by human melanoma cells in nude mice. The mechanisms by which tumor cells metastasize to bone are not well understood. We have investigated the role of the basement membrane glycoprotein, laminin, in bone metastasis, since antagonists to laminin have been shown to inhibit the formation of lung metastases. We studied the formation of osteolytic metastases caused by a human tumor which is known to cause osteolysis and hypercalcemia in nude mice. We found that tumor-bearing nude mice developed hypercalcemia, cachexia, and characteristic osteolytic lesions throughout the skeleton after injection of this human melanoma cell line (A375) into the left ventricle. When we gave injections to nude mice with A375 cells which had been exposed to C(YIGSR)3-NH2, a laminin-derived synthetic peptide containing three linear sequences of YIGSR with an amino-terminal cysteine which competes with laminin for its receptor, we found a decrease in the formation of detectable osteolytic bone metastases. The tumor cells were incubated with the antagonist and then inoculated into nude mice which were administered the antagonist i.p. Hypercalcemia and cachexia were also decreased in tumor-bearing mice treated with the laminin antagonist. In contrast, laminin itself increased the number of osteolytic bone metastases, as has been shown for other tumor cells. These data suggest that laminin plays a role in the formation of osteolytic bone metastases in this model and that laminin antagonists may be useful in the prevention of bone metastases in some human tumors.


Activation of omental milky spots and milky spot macrophages by intraperitoneal administration of a streptococcal preparation, OK-432. Omental milky spots are omentum-associated lymphoid tissues that cede peritoneal macrophages and participate in the immunity of the peritoneal cavity. We studied the changing surface features of milky spots and milky spot macrophages of Wistar rats, following the i.p. administration of OK-432, a killed streptococcal preparation (1 Klinische Einheit (unit) in 5 ml of phosphate-buffered saline) by the use of scanning electron microscopy. OK-432-activated macrophages demonstrated marked surface membrane activity and migrated through the stomata of the milky spot into the peritoneal cavity. The characteristic features of activated milky spots and milky spot macrophages were noted as early as 3 h following the administration of OK-432, and continued to be observed until 7 days after the injection. By 14 days after the injection, the structural integrity of the milky spot was partially lost. The activation of milky spots and milky spot macrophages by OK-432 provides a convenient in vivo system for the monitoring and study of i.p. cellular events.


bcl-2 gene transfer increases relative resistance of S49.1 and WEHI7.2 lymphoid cells to cell death and DNA fragmentation induced by glucocorticoids and multiple chemotherapeutic drugs. The S49.1 and WEHI7.2 murine lymphoid cell lines have been used extensively as models for investigations of programmed cell death ("apoptosis") induced by glucocorticoids such as dexamethasone. Infection of these thymus-derived T-cell lines with a recombinant retrovirus encoding the human M(r) 26,000 Bcl-2 oncoprotein resulted in marked resistance to DEX-mediated cell death and DNA degradation into oligonucleosomal fragments, without interfering with the ability of dexamethasone to suppress cellular proliferation and without lowering levels of glucocorticoid receptors. In contrast, high levels of p26-Bcl-2 production did not block cell killing and DNA fragmentation induced by H2O2, suggesting that the Bcl-2 impairs some but not all pathways for cell death in S49.1 and WEHI7.2 cells that are associated with the DNA fragmentation pattern typical of apoptosis. S49.1 and WEHI7.2 cells infected with bcl-2 but not control retrovirus also exhibited increased resistance to cell killing and DNA fragmentation induced by a wide variety of reagents, including the calcium ionophore ionomycin, the phorbol ester tetradecanoylphorbol acetate, the dihydrofolate reductase inhibitor methotrexate, the antimetabolite 1-beta-D-arabinofuranosylcytosine, and the microtubule inhibitor vincristine. These findings provide evidence that p26-Bcl-2 interferes with a pathway for cell death that is activated by multiple drugs used for the treatment of cancer.


In vivo and in vitro characteristics of interleukin 6-transfected B16 melanoma cells. Interleukin 6 (IL-6) is a multifunctional cytokine important in the inflammatory response. Its potential role as an antitumor agent has been suggested by its demonstrated activity in a variety of tumor models. The mechanism of antitumor activity has been proposed to be its enhancement of cytotoxic T-cell function. In the current work we demonstrate clear antitumor activity for this cytokine in a nonimmunogenic tumor system. B16 melanoma cells transfected with the human IL-6 complementary DNA demonstrated slower tumor growth in vivo. Tumors that developed from these cells had a prominent stromal matrix, an easily recognized infiltration of inflammatory cells, fewer mitotic figures, and fewer blood vessels. These in vivo findings corresponded with a greater adhesion of the IL-6-transfected B16 cells to stromal matrix proteins (laminin, fibronectin, and vitronectin) and a less prominent vascular response in an intradermal angiogenesis assay. Therefore, we propose that with weakly antigenic tumors, such as B16 melanoma, IL-6 may mediate important antitumor responses by nonspecific proinflammatory mechanisms.


An ovarian tumor marker with homology to vaccinia virus contains an IgV-like region and multiple transmembrane domains. The monoclonal antibody OVTL3 has a highly restricted reactivity with ovarian carcinomas and defines a surface glycoprotein, OA3, which has been used for immunotargeting. To understand why OA3 is found on ovarian tumors we isolated a complementary DNA by expression cloning. The clone encodes a 323-amino acid protein with 5 putative membrane spanning domains, reminiscent of a membrane receptor or channel, but of a new type with little or no sequence similarity with these families of proteins. Interestingly, the OA3 sequence is highly related to a vaccinia virus encoded protein (VA38) and its extracellular domain is a member of the immunoglobulin V region superfamily.


Non-Hodgkin's lymphoma time trends: United States and international data. Incidence data from the Surveillance, Epidemiology, and End Results Program of the National Cancer Institute, earlier incidence surveys, and the International Agency for Research on Cancer, mortality data from the National Center for Health Statistics, and population data from the Census Bureau were used to assess rates of non-Hodgkin's lymphoma. Mortality and incidence rates have been increasing for many years. Larger increases among older persons suggest a role for improving diagnosis, particularly during the 1950s and 1960s. Urban/rural and socioeconomic differences have diminished over time. Since the early 1970s, incidence rates increased at 3-4%/year, more rapidly than for all other cancers except melanoma of the skin and lung cancer among women. Incidence rates increased over all ages except the very young, among whites and blacks, in geographic areas both in the United States and internationally, and both sexes. During the 1980s, the impact of AIDS is apparent among young and middle-aged men. Differences in non-Hodgkin's lymphoma rates persist between races and sexes. Increases have been more marked for extranodal disease, particularly those arising in the brain, and for high-grade tumors. Explanations accounting for all the increases in rates are not readily available.


Changes in the descriptive epidemiology of non-Hodgkin's lymphoma in Great Britain? The problems associated with a diagnosis of non-Hodgkin's lymphoma are reviewed in the context of an apparent rise in incidence. It is concluded that a true rise exists and that further work is required to fully understand the significance of this.


Time trends of non-Hodgkin's lymphoma: are they real? What do they mean? Factors that need to be considered in the analysis of time trends in disease incidence are age, year of diagnosis, and birth cohort. When these are included in a log-linear model, a nonidentifiability problem arises from the linear dependence among these three time factors so that only specified functions of the parameters can be unambiguously determined. One of these invariant functions is the drift or the sum of the period and cohort trend. Non-Hodgkin's lymphoma incidence rates from Connecticut for the period 1935-1989 were analyzed for males and females. In addition to an age effect, both period and cohort significantly improved the fit of the model. The estimated drift shows that there has been a 10.3% increase in risk every 5 years since 1965 for females and 9.2% for males. It is unlikely that a trend of this magnitude can be attributed entirely to data artifact.


An overview of the classification of non-Hodgkin's lymphomas: an integration of morphological and phenotypical concepts. An analysis of trends in the incidence of non-Hodgkin's lymphoma requires an understanding of individual disease entities within this broad group. The non-Hodgkin's lymphomas represent a diverse group of malignancies that have in common an origin from lymphoid cells. Nevertheless, these disorders are heterogeneous in their clinical behavior, morphological appearance, cellular origin, etiology, and pathogenesis. A modern classification of non-Hodgkin's lymphomas must include an integration of morphological, immunophenotypical, and molecular concepts in order to delineate individual diseases within this broad group. Existing classification schemes such as the working formulation, while they may be useful in providing a guide to clinical management, cannot provide this information in the absence of other data. This point is most readily made with the low-grade B-cell lymphomas which include follicular lymphomas, mantle cell lymphomas, small lymphocytic lymphomas, immunosecretory disorders, and lymphomas of mucosa-associated lymphoid tissues. Each of these malignancies has a distinct phenotype and genotype, and indubitably each has a different etiology. The postthymic T-cell tumors are equally diverse. Analysis of epidemiological data from cancer registries must include a recognition that our ability to recognize individual diseases from historical data is limited. Studies of trends in the non-Hodgkin's lymphomas should attempt to delineate biological markers that may be of relevance to pathogenesis in both historical and prospectively accrued cases.


Changes in diagnosis of non-Hodgkin's lymphomas over time. The question is raised as to whether changes in criteria for the diagnosis of non-Hodgkin's lymphomas, both clinical and pathological, have changed over the past four decades in sufficient scale to create a spurious increase in the apparent incidence of this grouping of disease. In the decade of the 1970s refinement in histomorphological criteria for the diagnosis of Hodgkin's disease resulted in as many as 10-15% of cases which previously would have been diagnosed as Hodgkin's disease being diagnosed instead as non-Hodgkin's lymphoma. Other considerations, including distinction of non-Hodgkin's lymphomas from leukemias and plasma cell myelomas, and recent recognition of angioimmunoblastic lymphadenopathy and extranodal "pseudolymphomas" as variant forms of non-Hodgkin's lymphoma, appear to have added only marginally to the total of reported cases. It is concluded that the increase in reported incidence of non-Hodgkin's lymphoma cannot be explained on the basis of changes in diagnostic criteria.


Pathological classification of non-Hodgkin's lymphoma for epidemiological studies. Non-Hodgkin's lymphoma (NHL) consists of a heterogeneous group of disorders which have been difficult to study by epidemiological means in the past. However, recent advances in knowledge of the biology of NHL and improvements in its classification will greatly improve the quality of epidemiological studies in the future. Use of the Working Formulation and the current International Classification of Diseases for Oncology, along with paraffin immunohistochemistry, allow the delineation of NHL subgroups with possible etiological significance based on the biology of the disease. The collaboration of epidemiologists with expert pathologists in the design, performance, and evaluation of epidemiological studies of NHL is essential for such studies to be meaningful.


Familial aggregation of hematopoietic malignancies and risk of non-Hodgkin's lymphoma. Examination of risk factors that may be responsible for the increasing incidence of non-Hodgkin's lymphoma (NHL) over the past few decades would be incomplete without considering familial aggregation of hematolymphoproliferative neoplasms and the relative contributions of heredity and environment to the etiology of NHL. Reports of families with two or more NHL cases and sometimes additional members affected by other hematopoietic malignancies (multiple-case families) are summarized, as are findings from surveys and quantitative risk estimates from population-based studies of familial aggregation. The notable occurrence of various immunological abnormalities among multiple-case family members with and without NHL or related neoplasms is underscored, as is the diversity of types of other lymphoproliferative and hematopoietic malignancies among close relatives in these families. Preliminary evidence suggesting that multiple-case families may be more susceptible to certain environmental exposures is presented. An international registry of such families (particularly those identified in population-based studies) is proposed to clarify the interrelationship of genetic, familial, and environmental factors in the etiology of NHL.


Immunosuppressive therapy and acquired immunological disorders. Impairment of the immune system by drugs, such as azathioprine and cyclosporin, or by diseases, such as AIDS, represents the most firmly established cause of non-Hodgkin's lymphoma (NHL). Neither drugs nor diseases, however, can explain the increases in the incidence of NHL in the general population, for these include cohorts relatively unexposed to immunosuppressive drugs or to AIDS. Furthermore, no immunological disorder that is associated with an increased risk of NHL is known to have increased in incidence. Among alternative explanations is the possibility of increased exposure to a lymphomagenic agent in the environment that acts by nonimmunological means. Pesticides and herbicides may belong to this category, but they will not explain the substantial increases in NHL in urban populations. However, the evidence of an underlying viral aetiology for the lymphomas in several different forms of immune impairment may be relevant to the increases in NHL in the general population. Paralytic poliomyelitis and mumps represent examples of diseases that have changed their pattern of occurrence in this century, consequent on changes in social conditions. It is therefore not impossible that changes in hygiene and in population density have altered the average age of exposure to a virus, thereby increasing the likelihood of a lymphomagenic effect.


Lymphoma risks in populations with altered immunity--a search for mechanism. There have been numerous studies of lymphoma incidence in patient populations having diseases or taking medications that result in altered immunity. While many of these have uncovered substantially elevated risks, little has been done to use these observations to gain insights into the mechanisms of lymphomagenesis. Speculation about mechanism has centered on either immunosuppression or immunostimulation. The patterns of risk among kidney transplant recipients (higher risks for those receiving cadaveric grafts, having multiple transplants, or having received transplants in the earlier years of transplantation) favor the immunostimulation hypothesis. Likewise, the higher lymphoma risk associated with indices of increasing severity of disease among patients with sicca syndrome also support the role of immunostimulation over that of immunosuppression. However, an ordering of many of the more extensively studied conditions with altered immunity on the basis of the relative risk of lymphoma associated with these conditions reveals a pattern of risk which is not completely consistent with the level of either immune suppression or immune stimulation. Perhaps we have reached a point where epidemiologists working with laboratory and clinical immunologists can discern a more sophisticated underlying mechanism than the crude concepts of immunosuppression or immunostimulation that would explain the markedly differing lymphoma risks seen in various groups with marked immune abnormalities. If so, markers of more subtle alterations in this mechanism might be profitably explored for their role in lymphoma in general and as a tool to define the environmental causes of the epidemic increases.


Viruses other than HIV and non-Hodgkin's lymphoma. There are several viral infections which are known to cause lymphoma among animals; all establish latency in lymphoid cells. The human T-lymphotropic virus type I is a human virus which causes lymphomas among a subset of carriers. However, this virus is very restricted in its distribution and as such, is unlikely to play a role in the increase of non-Hodgkin's lymphoma (NHL). A highly prevalent infection, the Epstein-Barr virus (EBV) is known to play a role in the etiology of NHL among persons with acquired or inherited immune suppression. However, whether it is involved with "spontaneous" NHL is unknown. We have found evidence that among a group of 104 NHL patients with blood samples taken several years before diagnosis, there was an alteration in the antibody profile against the EBV which is quite similar to that seen for immune-suppressed patients prior to their diagnosis. This pattern is most evident in the oldest patients. This suggests that there may be an age-related subclinical immune suppression leading to chronic activation of EBV. If a viral infection is a major factor in the recent increase in NHL in the world, then we should consider the role of immune-suppressive exposures which have become widespread in recent decades.


Pesticides and non-Hodgkin's lymphoma.

Pesticides and non-Hodgkin's lymphoma. The incidence of non-Hodgkin's lymphoma (NHL) has increased over 50% in the last 15 years. This paper reviews the possible role of pesticides in this increase. While small increases in risk of NHL among farmers have been observed in general occupational surveys, recent studies focusing on specific pesticides have observed much larger risks. Frequent use of phenoxyacetic acid herbicides, in particular, 2,4-dichlorophenoxyacetic acid, has been associated with 2- to 8-fold increases of NHL in studies conducted in Sweden, Kansas, Nebraska, Canada, and elsewhere. Canine malignant lymphoma has also been associated with dog owner use of 2,4-dichlorophenoxyacetic acid and commercial lawn pesticide treatments. There are much fewer data linking NHL to other types of pesticides, but triazine herbicides, organophosphate insecticides, fungicides, and fumigants have also been associated with increased risk of NHL. Pesticide exposures are not limited to agricultural populations but are widespread in the general population through use on lawns, golf courses, rights-of-way, and elsewhere. Since the use of pesticides, particularly phenoxy herbicides, has increased dramatically preceding and during the time period in which the incidence of NHL has increased, they could have contributed to the rising incidence of NHL.


Radiation and non-Hodgkin's lymphoma.

Radiation and non-Hodgkin's lymphoma. Lymphomas are rarely, if ever, found to be in excess following exposure to ionizing radiation. Hodgkin's disease has never been linked to radiation, and the evidence for non-Hodgkin's lymphoma (NHL) is very weak. Low doses of radiation from diagnostic X-ray procedures or from occupational exposures do not appear to cause NHL. Mortality studies of atomic bomb survivors in Japan and other epidemiological studies with quantitative estimates of radiation dose also fail to find dose-response relationships. NHL may arise infrequently following high-dose, possibly near lethal, radiation treatments. Immunosuppression associated with the disease being treated, such as Hodgkin's disease, may contribute to the development of NHL. If radiation does not cause NHL, at least not by its accepted mechanism of action of breaking chromosomes, creating rearrangements, gene deletions, and mutations, perhaps other environmental mutagens and clastogens should not be considered likely causes of NHL.


Nutritional factors and the development of non-Hodgkin's lymphoma: a review of the evidence. Lymphomas of the non-Hodgkin's type represent a heterogeneous group of tumors, probably comprised of groups of related diseases each with a distinct etiology. The epidemiology of non-Hodgkin's lymphoma is poorly characterized, and little is known about factors which increase a person's risk of developing one of these tumors. Available epidemiological evidence suggests the influence of a number of environmental factors. Although diet is potentially one of the most significant environmental factors that could be related to disease etiology, very little has been done to investigate the role of diet in the development of non-Hodgkin's lymphoma. In this paper, the existing epidemiological findings are reviewed, and the plausibility of an etiological association between dietary factors and non-Hodgkin's lymphoma is considered in the context of relevant animal research.


Increasing incidence of non-Hodgkin's lymphoma: occupational and environmental factors. The incidence of non-Hodgkin's lymphoma (NHL) has been increasing steadily for the last 30 years, and attention is being focused on the possible causes of this increase. Possible explanations have included the exposure to viruses, radiation, nutrition, and pesticides, and these issues are addressed by other presentations in this workshop. The interest in a possible role of pesticides stems from the observation that farmers have an increased risk of NHL. However, farmers may also be exposed to oncogenic viruses carried by farm animals, and studies of abattoir workers and meat inspectors have found increased risks of NHL; although these findings are unlikely to be directly relevant to the general population, they do complement other suggestions that exposure to oncogenic viruses may be a factor in the general increase in NHL. Farmers may also be exposed to chronic antigenic stimulation which may increase the risk of NHL. This latter observation is consistent with the observation that NHL is associated with several autoimmune diseases which involve chronic antigenic stimulation. NHL has also been associated with a number of occupational exposures but these are generally rare and the findings are inconsistent, although a number of studies have found an increased risk of NHL in work involving exposure to wood, solvents, or related chemicals. Perhaps the strongest evidence of an association with an environmental exposure comes from two studies showing that use of hair dyes increases the risk of NHL. This exposure is relatively common in women, and hair dye use may account for approximately 20% of all NHL cases in women.(ABSTRACT TRUNCATED AT 250 WORDS)


Comments on occupational and environmental factors in the origin of non-Hodgkin's lymphoma. The review of the literature regarding non-Hodgkin's lymphoma and occupational and environmental factors presented at this workshop suggested associations with viruses, solvents, and hair dyes. A population-based case-control study among men from Iowa and Minnesota notes similar associations. Workers engaged in metal working, hair care, painting, and dry cleaning experienced nonsignificant excesses. Risks from specific exposures showed some variation by histological type. Both follicular and diffuse non-Hodgkin's lymphoma were associated with benzene. The diffuse type was linked to solvents other than benzene and formaldehyde, while the follicular was excessive among workers exposed to oils and greases.


Non-Hodgkin's lymphoma and occupational exposure. A case-control study was conducted to assess the effect of occupational exposures on the risk of non-Hodgkin's lymphoma. Interviews were conducted with 303 persons with non-Hodgkin's lymphoma newly diagnosed from January 1, 1980, to May 31, 1982, among residents of the Boston, MA, metropolitan area and 303 age and gender matched controls. The study found an increased risk of disease among persons employed in the agriculture, forestry, and fishing industry [relative risk (RR) = 3.0]; the construction industry [RR = 2.1]; and the leather industry [RR = 2.1]. The particular job groupings at increased risk were plant farmers and gardeners (RR unbounded); painters and plasterers (RR = 6.0); and carpenters, brick and stone masons, plumbers, and roofers (RR = 12.0). Although other exposures may have led to these increased risks, the findings in this study are consistent with an increased risk of non-Hodgkin's lymphoma for workers who may be exposed to chlorophenols or phenoxyacetic acids.


Prior medication use and health history as risk factors for non-Hodgkin's lymphoma: preliminary results from a case-control study in Los Angeles County. To determine whether non-Hodgkin's lymphoma (NHL) is related to prior medication use or health history, a population-based case-control study was conducted. A total of 619 male and female residents of Los Angeles County who were diagnosed with NHL between January 1, 1979, and June 30, 1982, were compared to individually age-, race-, and sex-matched neighborhood controls with regard to history of use of 49 different medications, 47 chronic and infectious diseases or other conditions, 15 types of immunizations, and 15 specific allergic reactions. Based on preliminary analyses, long-term regular use of aspirin and other pain relievers and greater than or equal to 2 mo of treatment with penicillin and other antibiotics were associated with significantly increased risk of NHL. Other drugs associated with greater risk of NHL were use of digitalis and estrogen replacement therapy by women, use of corticosteroids, and greater than or equal to 2 mo of use of tranquilizers. NHL was strongly associated with a prior history of cancer. Cases more frequently reported histories of kidney infections and anemia than did controls; a history of eczema appeared to be protective against NHL. Women who had been immunized against polio by injectable vaccine were at significantly lower risk of NHL than women who had not received this immunization. Among men, cholera immunization and allergy to nuts and berries were significantly protective. Subjects who had received a yellow fever immunization also had lower NHL risk. Further analyses of these data will attempt to establish the relative importance of these potential risk factors and to determine whether any are markers of early symptoms of NHL.


Does chronic fatigue syndrome predispose to non-Hodgkin's lymphoma? Chronic fatigue syndrome, an illness that frequently is associated with abnormalities of cellular immunity, has been reported anecdotally to be associated with an increased incidence of lymphoid hyperplasia and malignancy. This report describes an initial analysis of population-based cancer incidence data in Nevada, focusing on the patterns of non-Hodgkin's lymphoma prior to and subsequent to well described, documented outbreaks of chronic fatigue syndrome during 1984-1986. In a study of time trends in four age groups, the observed time trends were consistent with the national trends reported in the Surveillance, Epidemiology, and End Results Program. No statistically significant increase attributable to the chronic fatigue syndrome outbreak was identified at the state level. Additional studies are in progress analyzing the data at the country level, reviewing patterns in other malignancies, and continuing to monitor the cancer patterns over subsequent years.


Pathogenetic mechanisms in B-cell non-Hodgkin's lymphomas in humans. A very large proportion of non-Hodgkin's lymphoma in the United States are of B-cell origin. This group of tumors includes a variety of different pathological and clinical types. Chromosomal rearrangements play an important role in the pathogenesis of many of these tumors. In B-cells these translocation processes appear to develop as illegitimate products of physiological V-(D)-J or heavy chain switch rearrangements. The biology of the well-known chromosomal translocations is discussed. Additional biological factors in lymphomagenesis (aging, immunodeficiency, role of antigenic stimulation, and genetically determined susceptibility) are discussed.


Molecular basis of lymphomagenesis.

Molecular basis of lymphomagenesis. Lymphoid neoplasms, like all malignant tumors, arise as a consequence of the accumulation, in a single cell, of a set of genetic lesions that result in altered proliferation or increased clonal life span. The most frequently observed genetic abnormalities among the malignant non-Hodgkin's lymphomas are translocations, which appear to be lineage and, to a large extent, lymphoma specific. Recombinases that normally mediate the process of antigen receptor gene rearrangement appear to have an important (but not exclusive) role in the mediation of these translocations and of other types of gene fusion (e.g., deletion of intervening DNA). Frequently, such fusions result in the increased or inappropriate expression of crucially important proteins, many of which are transcription factors that regulate the expression of other genes. These abnormalities, however, do not appear to be sufficient to induce lymphoma, and it is likely that the additional genetic lesions required differ from one tumor to another. The likelihood of any given clone of cells accumulating a sufficient number of relevant genetic lesions to give rise to a lymphoma is probably a function of its life span. Prolonged survival of a cell clone may be mediated by viral genomes (e.g., Epstein-Barr virus and human T-cell leukemia/lymphoma virus type 1), by the abnormal expression of cellular genes that inhibit apoptosis (e.g., bcl-2), or by the mutation or deletion of cellular genes that are necessary for apoptosis, e.g., p53. The background rate at which genetic lesions occur is amplified by the interaction of inherited and environmental factors, the latter appearing to be the major determinant of incidence rates. However, inherited factors that influence lymphomagenesis, including variability in the ability to repair DNA damage or in the fidelity of antigen receptor recombinases for their signal sequences, may be crucial determinants of which particular individuals in a given environmental setting develop lymphoma.


Characterization of chromosome 11 translocation breakpoints at the bcl-1 and PRAD1 loci in centrocytic lymphoma. The chromosome 11q13 bcl-1 locus is rearranged in the majority of centrocytic lymphomas, a CD5-positive B-cell non-Hodgkins lymphoma, as a result of reciprocal translocation with the 14q32 immunoglobulin heavy chain genes. Although several 11q13 bcl-1 breakpoint sites have been characterized, a postulated bcl-1 oncogene was not identified. Recently, however, a gene encoding cyclin D1, designated PRAD1, was proposed as a candidate bcl-1 oncogene; accumulated evidence now indicates this gene is bcl-1. To further characterize 11q13 breakpoints in B-cell neoplasms, we analyzed 26 centrocytic lymphomas and 68 other B-cell cancers by Southern blot using a panel of breakpoint probes spanning 110 kilobases of the bcl-1 and PRAD1 loci. Nineteen centrocytic cases (73%) showed rearrangement, 15 at bcl-1 breakpoint sites and 5 at PRAD1 sites. One case was rearranged at both bcl-1 and PRAD1 loci. All but the latter case showed comigration of rearranged bcl-1 or PRAD1 bands and immunoglobulin heavy chain joining gene bands, consistent with the t(11;14). bcl-1 rearrangement was present in only one of 68 noncentrocytic B-cell neoplasms; none showed PRAD1 rearrangement. Thus, bcl-1 and PRAD1 rearrangement is strongly associated with centrocytic lymphoma, providing a useful molecular marker for classifying this subtype of lymphoma and suggesting an important role for PRAD1 cyclin D1 in the pathogenesis of this neoplasm.


A measure of genomic instability and its relevance to lymphomagenesis. A recent pilot study that we performed on 12 individuals who are involved in the cultivation and processing of grains and legumes suggests to us that we may have in hand a relatively quick, inexpensive, and highly sensitive assay that identifies individuals at increased risk for the development of lymphoid malignancy. The generation of this assay evolved from our interest in the causes and consequences of lymphocyte-specific chromosomal aberration.


Antigen selection in human lymphomagenesis. Although surface immunoglobulin plays a central role in the differentiation and growth of normal B-cells, its role in the growth of human B-cell malignancies is largely a matter of conjecture. Human follicular lymphomas are attractive systems to study in part because they are clones of cells sharing many similarities with germinal center B-cells which are critically dependent on antigen selection for survival. Nucleotide sequence information was determined for the immunoglobulin heavy chain variable genes expressed by two cases of follicular lymphoma. In addition, the germ line variable gene counterparts were also cloned and sequenced from biopsy material obtained from both of these patients. Numerous mutations from germ line were present in the variable genes from both of these cases, many of which accumulated during expansion and growth of these lymphomas. Moreover, the mutations that accumulated during tumor expansion were distributed in a manner that almost certainly was dependent on positive selection presumably mediated by contact with an antigen. These data indicate that antigen selection is probably important for the growth and clonal evolution of follicular lymphomas.


Follicular dendritic cells: B-cell proliferation and maturation. Follicular dendritic cells (FDC) play a pivotal role in the initiation and maintenance of the secondary humoral immune response. FDC located in the follicles of secondary lymphoid tissues are part of a special mechanism that handles antigen in a specific antibody-rich environment and are integral members of the antigen transport cell-FDC-iccosome-B-cell axis that leads to the formation of germinal centers, antibody-forming cells, and memory B-cells. This review briefly outlines the basic biology of FDC function and provides a source of pertinent references.


The past is prologue: use of serum banks in cancer research. In this paper, we emphasize the uses of serum banks in cancer research. These include not only case/control studies but also prospective seroepidemiological studies in which the development of a serological marker, such as a viral antibody or viral antigen, can be correlated with the subsequent development of cancer in either an active surveillance program or the use of cancer registries or hospital records. Several different methods of application of the cohort technique are illustrated by studies of hepatitis B antigen and hepatocellular carcinoma and of Epstein-Barr virus in relation to African Burkitt's lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. Collections of sera done for one purpose can often be utilized for another purpose, if properly stored and documented. Two examples are tests for human T-cell leukemia virus, type 1, antibody from sera done for a health survey in Barbados approximately 8 years earlier and the use of data determined for a prospective study of the incidence of Epstein-Barr virus infection and infectious mononucleosis in West Point Cadets for psychological factors affecting the development of clinical illness among those infected. Archival materials, such as frozen tissues and paraffin sections, may also now be utilized for identifying genomes of potential oncogenic viruses by the polymerase chain reaction.


Applications of experimental techniques to epidemiological studies of non-Hodgkin's lymphoma: use of archival tissues. Archival tissues, particularly formalin-fixed paraffin-embedded tumors, have become increasingly valuable in studies of the etiology of cancer. In non-Hodgkin's lymphoma, subclassification of tumors by immunophenotyping and identification of oncogenic viruses has allowed more accurate interpretation of associated epidemiological information. One such example is adult T-cell leukemia/lymphoma, which is not a single histopathological entity and usually is associated with human T-cell lymphotropic virus, type I. In addition to confirming the diagnosis, the pattern of virus distribution, utilized recently in studies of Epstein-Barr virus and human herpesvirus-6-associated lymphoma, has suggested which tumors are more likely to have the virus playing a passenger role (virus detected in uninvolved tissues) and in which tumors the virus may have an etiological role (virus restricted to tumor cells). Preservation and cataloguing of tumors and relevant clinical and demographic data may play an increasingly important role in demographic studies.


Quantification of the impact of known risk factors on time trends in non-Hodgkin's lymphoma incidence. The incidence of non-Hodgkin's lymphoma among white men in the United States was measured as 6.9/100,000 person-years in 1947-1950 and as 17.4 in 1984-1988. We have estimated how much the known and suspected diagnostic and risk factors might have contributed to this apparent increase of 152%. Firm conclusions cannot be drawn without more data on risk and changes in prevalence, but a reasonable range of impacts can be constructed. After accounting for the likely effects of misdiagnosis of Hodgkin's disease as non-Hodgkin's lymphoma, of the acceptance of new entities of non-Hodgkin's lymphoma, of familial factors, of human immunodeficiency virus and other immunosuppressive conditions or drugs, and of occupation, we estimate that the percentage increase in incidence was still 80% among all males and 42% among those aged 0-64. An agent carrying a relative risk of 2.0 rising in prevalence from 0 to 42% would account for the latter rise. Diet, hair dyes, and general environmental exposures to pesticides may be contributing, but currently estimated risks and changes in exposure levels do not appear large enough to account for the residual rise. Among men aged 75-84, some of the residual rise of 109% probably is diagnostic, but only further research will clarify the issue.


The effect of an amino acid-lowering diet on the rate of melphalan entry into brain and xenotransplanted glioma. Melphalan (L-phenylalanine mustard, L-PAM, alkeran; molecular weight, 305,000) is transported across tumor cell membranes and the blood-brain barrier by the large neutral amino acid (LNAA) transport system. Normally, plasma LNAA levels are high enough and the affinity low enough that this system does not transport much melphalan into the brain. However, plasma amino acids can be reduced by fasting and protein-free diet. We used this method to reduce competition and to increase melphalan transport into brain tumors. In nude mice fasted for 12 h and then fed a protein-free diet for 2 and 6 h, mean plasma LNAA levels were 46% and 42% of control values. Nude mice with xenotransplanted D-54MG human gliomas were used to study tissue distribution and uptake kinetics of [3H]melphalan in a control group and a diet group (after a 12-h fast and 2 h of a 0% protein diet). The K1 (blood-to-tissue transfer constant) of melphalan, determined by graphical analysis and by nonlinear fitting to a 2-compartment model, was higher in the diet group in all tumor regions except the necrotic center of subcutaneous tumors; the increase was significant in the tumor periphery of brain and s.c. tumors. The ratio of K1s (diet to control) varied from 1.2 to 1.3 in brain tumors, 1.9 to 2.1 in subcutaneous tumors, and 1.8 to 3.1 in tumor-free brain. The apparent [3H]melphalan distribution space was significantly higher in the tumor periphery of both brain and subcutaneous tumors of the 15- and 30-min diet group. We also measured blood-brain barrier transport of [alpha-14C]aminoisobutyric acid and blood flow (with [131I]iodoantipyrine): the K1 of [alpha-14C]aminoisobutyric acid was 28.1 +/- 6.6 (SE) in brain tumors and 24.3 +/- 8.9 microliters/g/min in subcutaneous tumors. Blood flow was 58.2 --> 3.9 in brain tumors and 5.2 +/- 0.4 ml/100 g/min in subcutaneous tumors. Fasting, when combined with a protein-free diet, reduces plasma amino acid levels and thereby reduces competition between melphalan and LNAAs. This may increase the amount of melphalan that can enter a brain tumor without increasing the administered drug dose and suggests a therapeutic manipulation that can be used to increase the delivery of melphalan.


Modulation of protein kinase C-epsilon by phorbol esters in the monoblastoid U937 cell. Expression of protein kinase C-epsilon was examined in the human monoblastoid U937 cell. This cell type contained the alpha, beta, and epsilon isoforms of protein kinase C (PKC). While PKC-epsilon content was slightly higher in the cytosolic than in the particulate fraction, the amount contained in the particulate fraction was higher than the alpha and beta isoforms which were predominantly localized to the cytosol. After an acute exposure to tetradecanoyl-13-phorbol acetate (TPA), PKC-epsilon translocated to the particulate fraction. Acute or chronic exposure to ionomycin did not alter content of the epsilon isoform. Longer exposures to TPA decreased PKC-epsilon in both cellular fractions. PKC-epsilon displayed a similar sensitivity to TPA-induced down-regulation as did PKC-beta while PKC-alpha was more resistant to this effect. After a 72-h exposure to 0.1 nM TPA, increases in the alpha and beta isoforms but not in PKC-epsilon were observed. However, 1,25-dihydroxy vitamin D3 and dibutyryl cyclic AMP which induce U937 differentiation enhanced PKC-epsilon expression.


Antilymphoma activity of human gamma delta T-cells in mice with severe combined immune deficiency. Human Burkitt lymphoma (Daudi) cells grow as disseminated tumors in mice with severe combined immune deficiency (SCID) after either i.v. or i.p. injection. These cells are lysed in vitro by human V gamma 9/V delta 2 T-cells that recognize the groEL homologue on the Daudi cell surface. We report that both Daudi cell-stimulated peripheral blood mononuclear cells (Daudi-PBMC) containing 41-95% of V gamma 9/V delta 2 T-cells and V gamma 9/V delta 2 T-cell clones prolong the survival of SCID mice given inoculations of a lethal dose of Daudi cells. Groups of 6-8-week-old SCID mice were given inoculations i.v. or i.p. of 10(5) Daudi cells followed (through different injection sites) by: (a) 10(7) Daudi-PBMC; or (b) 10(7) unstimulated PBMC; or (c) 0.9% saline solution. All animals in groups (b) and (c) died of disseminated lymphoma, and their survival was significantly shorter than that of mice in group (a) (P < 0.001 for both i.v. and i.p. routes). Significant antitumor effects were also detected when Daudi-PBMC were injected 4 days before or 4 days after Daudi cells (P < 0.05). In vivo depletion of murine natural killer cells by anti-asialo GM-1 rabbit antiserum did not affect survival, suggesting that these cells did not contribute to lymphoma killing. Daudi-PBMC did not exert in vivo antitumor activity against the control Raji lymphoma. Mice receiving i.p. injections of Daudi cells followed by cytotoxic V gamma 9/V delta 2 T-cell clones also survived significantly longer (P < 0.05 for 3 different clones) than animals given Daudi cells alone or Daudi cells followed by noncytotoxic gamma delta T-cell clones. Our results indicate that this model system can be used for studies of human antilymphoma T-cell responses in vivo.


Genotoxicity of environmental agents in human mammary epithelial cells. Despite an increasing incidence of human breast cancer, its etiology remains unknown. Since some environmental chemicals are stored in human breast fat and are rodent mammary carcinogens, determining the genotoxic potential of environmental agents in this key target tissue is important. An assay was developed for detecting genotoxic activity, as unscheduled DNA synthesis (UDS), induced by chemicals and UV radiation in early passage cultures of normal human mammary epithelial cells (HMEC) derived from 5 different women. In order to measure UDS in culture, reduction in the percentage of cells in S-phase was accomplished either by depriving the cells of epidermal growth factor and bovine pituitary extract or by contact inhibition of growth. Cultures were incubated with test chemicals for 24 h in the presence of [3H]-thymidine. UDS was quantitated autoradiographically as net grains per nucleus (nuclear grains minus cytoplasmic background, population average) with > or = 6 net nuclear grains considered in repair for any individual cell. A positive response was observed with UV radiation, benzo(a)-pyrene, aflatoxin B1, ethylmethanesulfonate, 1,6-dinitropyrene, 2-acetylaminofluorene, and tobacco smoke condensate but not 7,12-dimethylbenz(a)anthracene or 2,3,7,8-tetrachlorodibenzo-p-dioxin. These results demonstrate that HMEC from all 5 women examined have the ability to metabolize a variety of environmental chemicals to DNA-reactive forms. Furthermore, some chemicals known either to cause mammary cancer in rodents or to be contaminants in human breast tissue are genotoxic in HMEC. A positive response in passage 9 cultures was observed only with direct acting agents, suggesting that HMEC may lose their metabolic capabilities in longer-term cultures. The HMEC UDS assay may be used to address the role of environmental agents in human breast cancer by determining whether chemicals are DNA reactive or metabolized to DNA reactive species in this critical target tissue.


DNA strand breaks and DNA cross-links in peripheral mononuclear blood cells of ovarian cancer patients during chemotherapy with cyclophosphamide/carboplatin. DNA strand breaks and DNA cross-links were detected in peripheral mononuclear blood cells of 15 ovarian carcinoma patients by alkaline filter elution. These patients received therapy with 600 mg/m2 of cyclophosphamide and 350 mg/m2 of carboplatin. Blood samples were taken a day before and 16 to 18 h after a therapy cycle. The patients showed an increased elution rate of 37% compared with that of healthy controls before the current cycle of chemotherapy, probably due to treatment in a previous cycle of therapy. The difference was statistically significant (P < 0.02; U test). At the end of the actual cycle of therapy an average acceleration of the elution rate of 157% was found compared with that of controls (P < 0.01; U test). Compared with the rate before the cycle of therapy, the mean elution rate after treatment was accelerated by 89% (P < 0.01; Wilcoxon test). The amount of DNA-protein cross-links was also increased after drug application. The individual patients showed different responses after drug intake. While some patients showed hardly any alteration in the elution rate, others showed an acceleration of up to 400%. Monitoring the course of disease in six of these patients indicated that a strong acceleration in the elution rate after drug application is possibly linked to the success of the chosen cancer treatment as measured by a decrease in the tumor marker CA12-5 to the normal level. In another investigation the group of patients who had received non-alkylating antineoplastic agents showed no increase in DNA strand breaks compared with untreated controls. Thus, monitoring DNA single-strand breaks in the peripheral mononuclear blood cells of patients can help to evaluate the efficiencies of the cancer treatment as a composite of individual differences in resorption, metabolic activation and detoxification, and possibly some constitutional aspects of drug resistance to cyclophosphamide/cisplatin and probably to several other alkylating antineoplastic drugs. This may help in choosing an effective drug and in adjusting the doses of these drugs individually in the chemotherapy of cancer.


Proposed role of phosphatidic acid in the extracellular control of the transition from G2 phase to mitosis exerted by epidermal growth factor in A431 cells. Epidermal growth factor (EGF) has been shown to cause an inhibition of A431 cells in G2 phase within approximately 10 min, i.e., shortly before mitosis (Kinzel et al., Cancer Res., 50: 7932-7936, 1990). This system has been used to study the proposed role phospholipid metabolites, particularly phosphatidic acid (PA), may play (Kaszkin et al., Cancer Res., 51: 4328-4335, 1991) in the extracellular control of cells at the physiological restriction site in G2 phase. A431 cells responded to EGF with a dose-dependent formation of phosphatidic acid (PA) which correlated with the dose-dependent G2 delay as well as with their time courses. The G2 delay induced by EGF as well as PA mobilization were effected in conditioned medium or in fresh medium containing bovine serum albimun instead of serum, i.e., under the conditions necessary for precursor studies to be carried out. The major pathway of PA formation was probably via phospholipase C-mediated breakdown of phosphatidylinositol and diacylglycerol kinase: (a) the dose response of PA formation correlated with that of total inositol phosphate accumulation; (b) little diacylglycerol was found and then only at a high EGF concentration; (c) prelabeling with [1-14C]arachidonic acid resulting in a large specific labeling of phosphatidylinositol led to an EGF-induced, dose-dependent formation of radioactive arachidonyl-PA (correlated with that of total PA and inositol phosphate), but in the presence of a primary alcohol not to the formation of radioactive phosphatidylalcohol; (d) prelabeling with [1-14C]oleic acid led to the EGF-induced formation of labeled PA, which in the presence of a primary alcohol was only slightly reduced to the advantage of very low levels of labeled phosphatidyl alcohol, thus demonstrating that an EGF-effected activation of phospholipase D did occur but contributed little to the general PA level. An alternative mobilization of PA was attempted with the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA), which was shown to activate phospholipase D in A431 cells and to elicit PA from a phospholipid pool which was not significantly labeled with radioactive arachidonic acid. The TPA-induced degree of PA formation and of the G2 delay correlated. Both phenomena were considerably larger with fresh medium containing 0.5% bovine serum albumin instead of serum than in conditioned medium.(ABSTRACT TRUNCATED AT 400 WORDS)


Chemoprevention of colon carcinogenesis by the synthetic organoselenium compound 1,4-phenylenebis(methylene)selenocyanate. The chemopreventive effect of 40% and 80% maximum tolerated dose (MTD) levels of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) administered in the diet during the initiation phase (2 weeks before, during, and up to 3 days after carcinogen administration) and the post-initiation phase (3 days after carcinogen treatment until termination) of azoxymethane (AOM)-induced colon carcinogenesis was studied in male F344 rats. The MTD of p-XSC was determined in male F344 rats and found to be 50 ppm. Beginning at 5 weeks of age, all animals were divided into various experimental groups (42 rats/group) and fed the high-fat semipurified diet or diets containing 20 (40% MTD) and 40 (80% MTD) ppm p-XSC. At 7 weeks of age, all animals (30 rats/group) except the vehicle-treated groups (12 rats/group) were administered s.c. injections of AOM (15 mg/kg body weight/week for 2 weeks). Three days after the second injection of AOM or vehicle (normal saline), groups of animals fed the p-XSC diets and control diet were transferred, respectively, to control diet and p-XSC diets and continued on these diets until the termination of the study. All animals were necropsied during the 36th week after AOM treatment. Colonic mucosal prostaglandin E2 and selenium-dependent glutathione peroxidase were measured in animals fed the control and p-XSC diets at the termination of the study. The results indicate that 40 ppm p-XSC administered during the initiation phase significantly inhibited the colon tumor incidence (percentage of animals with tumors). Dietary p-XSC administered at 20 and 40 ppm levels during the initiation phase significantly inhibited colon tumor multiplicity (tumors/animal and tumors/tumor-bearing animal). Colon tumor incidence and multiplicity were significantly reduced in groups fed 20 and 40 ppm p-XSC diets at the postinitiation phase of carcinogenesis. Colonic mucosal selenium-dependent glutathione peroxidase activity was increased, and prostaglandin E2 was reduced in animals fed the p-XSC diet compared to animals fed the control diet. Whereas the precise mechanisms of p-XSC-induced inhibition of colon carcinogenesis remain to be elucidated, it is likely that the effect during the initiation and postinitiation phases may be due to alteration in carcinogen metabolism and to modulation of prostaglandin synthesis and selenium-dependent glutathione peroxidase activity.


Reversal of basic fibroblast growth factor-mediated autocrine cell transformation by aromatic anionic compounds. NIH-3T3 cells transfected with basic fibroblast growth factor (bFGF) fused to a signal peptide sequence (spbFGF cells) are transformed in vitro and tumorigenic in vivo. Treatment of spbFGF cells with low and nontoxic concentrations (0.5-2.5 micrograms/ml) of negatively charged, nonsulfated aromatic compounds (e.g., aurin tricarboxylic acid, 4-hydroxyphenoxyacetic acid) resulted in restoration of their normal proliferative rate, morphological appearance, and adhesion properties. Binding and cross-linking experiments using 125I-labeled bFGF revealed that these alterations were associated with an up-regulation of high affinity receptors bFGF receptors was induced by these compounds in spbFGF cells that were seeded on fibronectin to enforce a firm cell attachment and flattening. Thus, induction of spbFGF cell adhesion and spreading may not be related to restoration of normal bFGF-receptor interactions. Although the negatively charged aromatic compounds mimic many of the effects of heparin in other systems (e.g., release of heparin- and heparan sulfate-bound proteins, inhibition of heparanase), heparin, heparan sulfate, and dextran sulfate were not effective at the low concentrations of the anionic compounds used in the present study. Likewise, suramin, a sulfated aromatic molecule, was effective at toxic concentrations, 400-600-fold higher than the nonsulfated aromatic compounds. The development of defined, nontoxic anionic compounds may provide a new strategy to interfere with the autonomous and anchorage independent mode of cell growth involved in autocrine cell transformation and cancer.


Immunization of colorectal cancer patients with modified ovine submaxillary gland mucin and adjuvants induces IgM and IgG antibodies to sialylated Tn. Tn and sialylated Tn (sTn) are blood group-related epitopes expressed on mucins of colon carcinoma and other epithelial tumors and are, therefore, potential targets for immunological control. We have immunized 20 colorectal cancer patients at high risk for recurrence with a vaccine consisting of partially desialylated ovine submaxillary gland mucin (modified OSM) which contains both Tn and sTn determinants. Six patients were treated with modified OSM alone (group 1), eight patients were treated with modified OSM and the immunological adjuvant DETOX (group 2), and six patients were treated with modified OSM and Bacillus Calmette-Guérin (group 3). Pre- and postvaccination sera were tested by enzyme-linked immunosorbent assay and dot blot immune stains for antibodies reactive with modified OSM. Antibody titers increased in 4 of 8 patients immunized with modified OSM and DETOX, in 5 of 6 patients immunized with modified OSM and B. Calmette-Guérin, and in 0 of 6 patients receiving modified OSM without adjuvant. The specificity of induced IgM and IgG antibodies was confirmed by demonstrating reactivity with OSM, bovine submaxillary mucin, and synthetic glycoconjugates sTn-human serum albumin (HSA) and Tn-HSA in enzyme-linked immunosorbent assay and immune stains. Median IgM pre-postvaccination reciprocal titers were 20/80 for Tn-HSA and 10/320 for sTn-HSA. Low level IgG antibody titers against sTn-HSA were detected after vaccination in 7 patients. Toxicity was limited to inflammatory skin reactions at the site of vaccination resulting from the adjuvants. No inflammatory infiltrates were seen in the skin when the modified OSM vaccine was administered in the absence of an immunological adjuvant. These results demonstrate that sTn and Tn can be recognized by the human immune system and that vaccines containing these structures can be administered safely with immunological adjuvants. Attempts to augment the immunogenicity of these carbohydrate antigens by covalent attachment to immunogenic carrier proteins and the use of more potent immunological adjuvants are now being pursued.


Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells. The mutagenic spectrum induced by aflatoxin-DNA lesions in DNA repair deficient and repair proficient human cells was investigated. The reactive metabolite aflatoxin B1-8,9-epoxide was synthesized and reacted in vitro with the shuttle vector plasmid pS189. Plasmids were transfected into human fibroblasts and allowed to replicate, and the recovered plasmids were screened in indicator bacteria for plasmid survival and mutations in the supF marker gene. Sequence data were obtained from 71 independently arising mutants recovered from DNA repair deficient xeroderma pigmentosum (XP) cells [XP12BE(SV40)] and 60 mutants recovered from a DNA repair proficient cell line (GM0637). Plasmid survival was lower and mutation frequency higher with the XP cells, and the mutation hotspots differed substantially for the 2 cell lines. Most mutations (> 90%) were base substitutions at G:C pairs, only about one-half of which were G:C-->T:A transversions, the expected predominant mutation. One-third of the mutations at GG sites and none of those at isolated Gs were G:C-->A:T transitions. Tandem base substitutions also occurred only at GG sites and were found only with XP cells. The location of mutation hotspots with either cell line did not correlate with the level of modification within the sequence as assessed by a DNA polymerase stop assay. These results suggest that the DNA repair deficiency associated with XP can influence not only the overall frequency of mutations but also the distribution of mutations within a gene. The finding of transition mutations exclusively at GG sites may be of predictive value in attempts to link dietary aflatoxin exposure to cancers associated with specific mutations in the c-ras oncogene and the p53 tumor suppressor gene.


A region of antisense RNA from human p120 cDNA with high homology to mouse p120 cDNA inhibits NIH 3T3 proliferation. The human nucleolar p120 protein is a proliferation-associated antigen which is expressed in G1 and peaks during the early S phase of the cell cycle. Overexpression of the human p120 protein caused the transformation of NIH 3T3 cells and expression of an antisense p120 construct inhibited the growth of NIH 3T3 cells (Perlaky et al., Cancer Res., 52:428-436, 1992). The middle region of the antisense p120 RNA was found to be almost as inhibitory as the full length antisense construct but the 5' and 3' antisense portions did not affect NIH 3T3 cell proliferation. After the mouse p120 complementary DNA was cloned and sequenced, comparison with the human p120 complementary DNA showed a striking conservation of 85% of the nucleotide sequence and 96% of the amino acid sequence. The two ends of the p120 molecule had less homology in their nucleotide and amino acid sequences. Based on this homology, the observed inhibitory effects of the middle portion of antisense human p120 RNA may be related to suppression of mouse p120 expression by RNA:RNA duplex formation. The high evolutionary conservation of the middle region suggests it has a critical role for the function of this protein.


Lethality, DNA alkylation, and cell cycle effects of adozelesin (U-73975) on rodent and human cells. Adozelesin (U-73975) is an extremely potent cytotoxic agent which causes 90% lethality, after 2 h exposure in vitro, of Chinese hamster ovary and lung (CHO and V79), mouse melanoma (B16), and human ovarian carcinoma (A2780) cells at 0.33, 0.19, 0.2, and 0.025 ng/ml, respectively. Under similar conditions, Adriamycin and cisplatin had 90% lethality values in CHO cells of 150 ng/ml (= 249 nM) and 6800 ng/ml (= 2266 nM), respectively. The relative drug sensitivity of the cell lines (A2780 > V79, B16, CHO) was correlated to the relative amounts of [3H]adozelesin alkylated to DNA. The greater sensitivity of A2780 was due to (a) greater DNA alkylation at different drug doses and (b) greater intrinsic sensitivity of A2780 which resulted in greater cell kill at comparable DNA alkylation. Phase specific toxicity studies show that adozelesin was least lethal to CHO cells in mitosis and very early G1. Lethality increased as cells progressed through G1 and was maximal in late G1 and early S. Mitotic cells had lower drug uptake and correspondingly less drug binding to DNA than G1 or S-phase cells. However, based on the amount of drug alkylated per micrograms of DNA, cells in M, G1, and S were equally sensitive. Therefore, the lower sensitivity of M-phase cells was due to lower drug uptake. Adozelesin had three different effects on progression of CHO, V79, B16, and A2780 through the cell cycle: (a) slowed progression through S which resulted in significantly increasing the percentage of S-phase cells. This effect was transient; (b) cell progression was blocked in G2 for a long time period; (c) the response of the cell lines to the G2 block differed. CHO and V79 cells escaped G2 block by dividing and entered the diploid DNA cycle or did not undergo cytokinesis and became tetraploid. On the contrary, B16 and A2780 cells remained blocked in G2 and did not become tetraploid. Cell progression was inhibited in a similar manner when a synchronized population of M, G1, or S-phase cells were exposed to adozelesin.


Antitumor effects of a bispecific antibody targeting CA19-9 antigen and CD16. Bispecific murine monoclonal antibodies that target tumor and Fc gamma RIII (CD16) can promote relevant tumor lysis by large granular lymphocytes. For these antibodies to be clinically useful, their properties should be maintained in vivo, where competing human immunoglobulin, shed target antigen, and shed CD16 may be encountered. At a minimum, bispecific antibody antitumor effects should be preserved in whole blood. Furthermore, potentiation of tumor lysis should be reflected by demonstrating the ability of bispecific antibody-retargeted effector cells to infiltrate and mediate lysis of organized tumor. If these characteristics are demonstrated, and there is evidence of in vivo efficacy of bispecific antibody-based therapy in a relevant animal model, further clinical development of such antibodies would be warranted. In this report the ability of CL158 bispecific antibody supernatants to mediate lysis of SW948 tumor growing in monolayer is shown to be preserved in the presence of interleukin 2-activated whole blood. When SW948 cells were grown in vitro as multicellular human tumor spheroids, incubation with interleukin 2-activated lymphocytes (LAK cells) and CL158 led to structural and widespread necrosis. This was dependent on CL158 and resistant to competition by pooled human immunoglobulin or interleukin 2-exposed whole blood. These effects were not promoted by the monospecific antibodies produced by the parent clones of CL158 and were not observed when the IgG2a variant of CA19-9 antibody, which mediates conventional antibody-dependent cellular cytotoxicity, was used instead of its bispecific derivative. To examine the efficacy of bispecific antibody-based treatments on in vivo tumor, scid mice bearing early s.c. SW948 xenografts were treated with interleukin 2 for 5 consecutive days, supplemented by three i.v. injections of 10(7) human LAK cells and various antibodies. Treatment of mice bearing SW948 tumors with LAK cells did not retard tumor growth, but when CL158 was added, significant delays in tumor growth were observed. Tumor growth delay required treatment with both LAK cells and the bispecific antibody. Treatment with the IgG2a variant of CA19-9 antibody, alone or with LAK cells, had no effects on tumor growth. Although the mechanisms of these antitumor effects require further study, it is clear that human LAK cell treatment of animals bearing early, established s.c. tumors is enhanced by the addition of bispecific antibodies with relevant binding characteristics. When compared with the IgG2a isotype variant of CA19-9 monoclonal antibody, this bispecific antibody offers the advantages of preservation of activity in physiological conditions, infiltration and disruption of organized tumor in vitro, and antitumor effects in a relevant xenograft model.(ABSTRACT TRUNCATED AT 400 WORDS)


Synchronization of breast cancer cell proliferation in vivo by combined hormonal and polyamine manipulation. Optimal synchronization of breast cancer cell proliferation by hormonal means may be limited by cellular heterogeneity in sensitivity to the multistep activation of growth following initial hormone binding to the receptor. We hypothesized that induced synchronous growth may be improved by combined manipulation of the polyamine (PA) pathway since we have previously shown that PAs are distal effectors of hormonal action on proliferation in breast cancer. To test our hypothesis, we induced an initial phase of hormone and PA depletion (castration plus administration of the PA synthesis inhibitor alpha-difluoromethylornithine) in rats bearing N-nitrosomethylurea induced mammary tumors. This was followed by transition phase of hormone repletion in the presence of alpha-difluoromethylornithine (to push the cells into the proliferative cascade up to the distal step controlled by PA) and finally a phase of hormone and PA repletion. Simultaneously, groups of rats were subjected to hormone/PA depletion/repletion individually. The effects of these manipulations on the labeling indices (LIs) of glandular, myoepithelial, and nonepithelial cells were estimated by autoradiography. The combined hormone/PA manipulation yielded the highest degree of synchronization with LIs of the glandular and myoepithelial cells being approximately 2-fold over intact control after only 2 or 3 days of combined repletion. In contrast, hormone treatment alone restored the LIs of glandular cells only to control levels and minimally influenced those of myoepithelial cells. PA manipulation alone failed to affect the LIs of any cell type. Although the rate of tumor regrowth was highest with the combination treatment, the absolute tumor volumes did not differ significantly at the end of the repletion phase between the three regimens. These results indicate that combined hormone/PA manipulation provides the best "therapeutic window" (LI/tumor volume) for implementation of kinetically based cytotoxic chemotherapy.


Glycosylation pathways in the biosynthesis of gangliosides in melanoma and neuroblastoma cells: relative glycosyltransferase levels determine ganglioside patterns. In order to elucidate some of the factors that determine the characteristic expression of gangliosides in malignant melanoma and neuroblastoma the levels of ganglioside synthases (glycosyltransferases) were determined in a panel of cell lines from those tumors that exhibited a wide range of ganglioside composition. Sialyltransferases (GM3, GD3, GD1a, and GT1b synthases), N-acetylgalactosaminyltransferases (GM2 and GD2 synthases), and galactosyltransferase (GM1 and GD1b synthases) were analyzed in crude membrane preparations from these cells. The results confirmed the importance of GM3 and GD3 synthases in determining the prominence of the a (GM3 to GT1a) or b (GD3 to GQ1b) biosynthetic pathways. The overall ganglioside composition in cells was found to be dependent on the relative levels of specific enzymes acting sequentially or in competing pathways. In general, the pattern and levels of transferases correlated with the actual ganglioside content of the cell line, although several important discrepancies were noted. For example, in cell lines containing high amounts of GD2 ganglioside, the level of the preceding enzyme in the pathway (GD3 synthase) was unexpectedly low. Thus, the high GD2:GD3 ratios characteristic of most neuroblastomas result from low levels of GD3 synthase as well as high levels of GD2 synthase. In other cell lines, GD3 synthase was completely absent, resulting in the synthesis of GM2, but not GD2, by N-acetylgalactosaminyltransferase I, as would be expected. It was concluded that different glycosyltransferases play key roles in determining glycolipid expression in different cell types.


Incidence of c-Ki-ras activation in N-methyl-N-nitrosourea-induced mammary carcinomas in pituitary-isografted mice. We found previously that mouse mammary epithelial cells cultured in the presence of the mammogenic hormones progesterone and prolactin and treated with the carcinogen N-methyl-N-nitrosourea produced a high frequency of hyperplastic alveolar nodules and carcinomas with squamous metaplasia upon transplantation to syngeneic mice. The majority of these mammary transformants had an activated c-Ki-ras proto-oncogene with a specific point mutation in codon 12 (G35 to A35). To determine whether these in vitro findings parallel mammary carcinogenesis in vivo, virgin female mice were pituitary isografted to increase their circulating levels of progesterone and prolactin. The pituitary isograft results in an increase in proliferation, leading to lobulo-alveolar development and differentiation of the mammary epithelial cells. Five weeks after pituitary isografting, the mice were treated with a single injection of N-methyl-N-nitrosourea (50 micrograms/g body weight). Greater than 90% of the N-methyl-N-nitrosourea-treated mice developed mammary carcinomas between 3 and 7 months after treatment. The majority (75%) of the carcinomas had histopathology identical to that of tumors induced in vitro in the presence of progesterone and prolactin. A number of the mammary cancers (17%) induced in pituitary-isografted mice also had the identical point mutation in the c-Ki-ras proto-oncogene found in the in vitro studies. These results suggest that the hormonal milieu around the time of carcinogen exposure affects not only the incidence and phenotype of the mammary transformants but also the molecular events associated with mammary carcinogenesis.


Stage-specific expression of cancer-associated type 1 and type 2 chain polylactosamine antigens in the developing pancreas of human embryos. Expression of type 1 and type 2 chain carbohydrate antigens during the course of morphogenesis of human embryonic pancreas was investigated using specific monoclonal antibodies and compared with the carbohydrate antigen profiles of human pancreatic cancers. The type 2 chain antigens, such as stage-specific embryonic antigen 1 (Le(x)) and I-antigens, appeared much earlier than the type 1 chain antigens; the epithelial cells of primitive foregut were Le(x)+I-antigen- in the embryos at Carnegie stages 16-23, while the pancreatic primordial cells, which had differentiated from the Le(x)+ gut epithelial cells, were Le(x)-I-antigen+ at Carnegie stages 22-23. The type 1 chain antigens, such as Le(a), Le(b), Le(c), and their sialylated derivatives, were not expressed in any cells at these stages and appeared much later in the pancreas of the 10-12-week embryos, when the primitive pancreatic ductal cells in the primordia exhibited an extensive budding of the daughter cells. At this stage, Le(a) appeared and was expressed strongly in the epithelial cells of primitive pancreatic ducts as well as in the daughter cells that were destined to differentiate into future centroacinar cells; Le(b) was localized in the daughter cells which were to become future acinar cells; and Le(c) was specifically expressed in the daughter cells which were to form future Langerhans islets. With regard to the sialylated derivatives of Le(a), expression of the 2-3 sialyl Le(a) antigen was limited to the epithelial cells of the primitive pancreatic ducts, while the 2-6 sialyl Le(a) antigen was strongly expressed in the future centroacinar cells, which had differentiated from the corresponding daughter cells. Among these antigens, the Le(a) and 2-3 sialyl Le(a) antigens showed the highest incidence in human pancreatic cancer tissues. These results indicate that the expression of these carbohydrate antigens in embryonic pancreas is differentiation dependent and cell lineage specific and that most human pancreatic cancer cells mimic the carbohydrate antigen profile of the epithelial cells of the primitive pancreatic ducts in human embryos.


Estrogen receptor-directed radiotoxicity with Auger electrons: specificity and mean lethal dose. To assess the feasibility of using estrogen receptor-directed therapy with Auger electron-emitting ligands for therapy of estrogen receptor (ER)-containing cancers, we synthesized and evaluated the radiotoxicity of several 123I-labeled estrogens to specifically kill ER+ cells in culture. Auger electrons have been previously shown to be of short range, generally less than the dimensions of a cell, so that to use them therapeutically a mechanism is needed to deliver the Auger electron-emitting nuclide to the vicinity of the DNA. Since it is now well established that the estrogen receptor, when bound to estrogen, forms a high affinity association with distinct estrogen response elements in the DNA, we wished to test the hypothesis that a short exposure of cells to a 123I-labeled estrogen would be specifically radiotoxic to ER+ cells, and that the decays per cell needed for cell killing would be compatible with reasonable levels of receptor occupancy. Using the halodestannylation reaction with tributyl tin precursors of several estrogens and commercially available iodine-123, we prepared the iodoestrogens, E-17 alpha(-)[123I]-iodo-11 beta-methoxyestradiol and 2(-)[123I]iodo-1,1-bis(4-hydroxyphenyl)--2-phenylethylene, at high specific activities, in several cases at essentially the specific activity of 123I itself, 240,000 Ci/mmol. When various concentrations of either of the 123I-labeled estrogens were incubated for 1 h with a subline of ER+ Chinese hamster ovary cells and the washed cells plated for survival assays, a dose-dependent, unlabeled estradiol-inhibitable reduction in survival was observed. In contrast, Chinese hamster ovary cells not expressing estrogen receptor showed little sensitivity to the radiotoxicity of the 123I-labeled estrogens. Calculations based on the assayed residence time of the iodoestrogens in the cells indicate that several hundred decays per cell are sufficient to kill cells.


Perioperative immunotherapy with recombinant interleukin 2 in patients undergoing surgery for colorectal cancer. Major surgery impairs the cellular immune response. We have therefore studied the immunological effects of low-dose recombinant interleukin 2 given to patients undergoing surgery for colorectal cancer to determine whether this agent has potential in perioperative adjuvant immunotherapy. Patients were randomly allocated to control (n = 13) or treatment groups (n = 12). Immunological studies of both lymphocyte function and subset number were performed preoperatively and on Days 1, 4, 7, and 10. Treatment with recombinant interleukin 2 prevented the postoperative fall in both natural killer and lymphokine-activated killer cell cytotoxicity, clearly demonstrated in the control group. The treatment group also showed in vivo T-cell activation with an initial lymphopenia followed by a rebound lymphocytosis and upregulation of the subset markers CD25 (interleukin 2 receptor) and CD45RO (T-memory cells). These combined effects may have important consequences in controlling metastatic dissemination of tumor during the vulnerable perioperative period.


Cadherin dysfunction in a human cancer cell line: possible involvement of loss of alpha-catenin expression in reduced cell-cell adhesiveness. A human lung cancer cell line, PC 9, was analyzed to elucidate the molecular mechanisms of dysfunction of cadherin-mediated cell-cell adhesion in cancer. Although PC 9 cells strongly expressed E-cadherin at the cell membrane, which was indistinguishable immunochemically from functional E-cadherin, they did not show tight cell-cell adhesion and had reduced E-cadherin-mediated aggregation activity. Immunoprecipitation with E-cadherin and Western blot analysis revealed that PC 9 cells did not express alpha-catenin, a cadherin-associated protein, suggesting that this was the cause of the cadherin dysfunction in the cell line. In addition, Northern and Southern blot analyses disclosed homozygous deletion of part of the alpha-catenin gene, which might have resulted in the loss of alpha-catenin expression in PC 9 cells.


Phase II trial of suramin in patients with advanced renal cell carcinoma: treatment results, pharmacokinetics, and tumor growth factor expression. Twenty-six patients with advanced renal cell carcinoma were treated with suramin administered by continuous infusion, with dosing determined by a nomogram. One patient achieved a partial response and five patients achieved a minor response or had stable disease for > 3 months. Toxicities included an immune-mediated thrombocytopenia in one patient and Staphylococcus sepsis that was not associated with neutropenia in five patients. Pharmacokinetic parameters were determined by the ADAPT II MAP-Bayesian parameter estimation program. Patient data were fit using a two-compartment open model and first-order rate elimination. This showed a wide interpatient variation in time to target level (median, 13.8 days), volume of distribution (median, 15.2 liters/m2), and t1/2-beta (median, 20.6 days). The patients who achieved a partial response, minor response, or stable disease had a slower elimination rate of suramin, compared to patients with progressive disease. Tumor specimens were obtained prior to therapy and were analyzed for the production of five different growth factor-specific RNA transcripts. These included transforming growth factor alpha, acidic fibroblast growth factor, basic fibroblast growth factor, and platelet-derived growth factor types A and B. No difference in the pattern of growth factor expression was seen in tumors of responding and nonresponding patients. Suramin does not have significant antitumor activity in renal cell carcinoma. The wide variability in pharmacokinetics suggests that individual dosing should be used in future trials of suramin for treatment for other malignancies. Pertinent corollary studies of tumor biology and clinical pharmacology should be included whenever possible in clinical trials in patients with renal cell carcinoma.


Absence of HSP28 synthesis and phosphorylation during the development of chronic thermotolerance in murine L929 cells. We investigated the correlation between chronic thermotolerance development and phosphorylation, synthesis, or expression of the HSP28 family in murine L929 cells. Chronic thermotolerance developed during heating at 41.5 degrees C as indicated by a biphasic survival curve. However, heat-induced phosphorylation of HSP28 was not detected. Furthermore, we failed to detect HSP28 synthesis during chronic heating by using two-dimensional polyacrylamide gel electrophoresis. The lack of HSP28 synthesis was also confirmed in acute thermotolerance. Similar results were observed in NIH 3T3 cells. Although Southern blots clearly demonstrated the presence of the HSP28 gene in genomic DNA, Northern blots failed to demonstrate its expression. Unlike HSP28, the expression of constitutive and inducible HSP70 genes, along with the synthesis of their proteins, were stimulated during chronic heating at 41.5 degrees C in L929 cells. These results suggest that HSP28 synthesis and its phosphorylation are not required to develop both chronic and acute thermotolerance in L929 cells.


Inhibitory effect of 2-deoxy-D-glucose on liver tumor growth in rats. Since tumor cells are more dependent on glycolysis for energy supply than other cells, we tested whether its inhibition by 2-deoxy-D-glucose (2-DG) affects tumor growth. Male Wistar rats were inoculated in the liver with tumor cells from a chemically induced colonic adenocarcinoma. From day 5 after inoculation 2-DG (400 mg/kg/24 h) was continuously infused into the hepatic artery for 5 days; controls received saline in the same fashion. Seven days after the end of infusion, the animals were sacrificed. A second experimental group of rats was treated with isolated liver perfusion for 30 min with oxygenated blood through the portal vein and hepatic artery simultaneously. In the perfusate, 400 mg/kg 2-DG were added, and the rats were sacrificed at 10 days after perfusion. A first control group underwent perfusion without 2-DG, and a second control group received i.v. infusion of 2-DG (400 mg/kg/30 min) for 30 min over 5 days. A nontreated control group was also added. All animals survived the procedures. The concentration of blood glucose increased in the rats receiving 2-DG i.v. and intraarterially but was unchanged in the other groups. The tumor growth was significantly reduced by 2-DG in all experimental groups, with no difference between the groups. It is therefore concluded that 2-DG is of potential interest in the treatment of malignancies. Since local application of 2-DG avoids the risk for systemic side effects, this approach should be explored further.


Gene-specific oligonucleotide probes for alpha, mu, pi, and microsomal rat glutathione S-transferases: analysis of liver transferase expression and its modulation by hepatic enzyme inducers and platinum anticancer drugs. Glutathione S-transferases (GSTs) play an important role in the detoxification of diverse electrophilic chemicals, including anticancer drugs. Gene-specific oligonucleotide probes were developed to monitor the expression of individual GST mRNAs in livers of adult male rats treated with drugs and other chemical modulators of GST expression. Northern blot analysis of total liver RNA using probes specific for individual GSTs belonging to classes alpha (GSTs Ya1, Ya2, Yc), mu (GSTs Yb1, Yb2, Yb3), pi (GST Yp), and GSTms demonstrated the expression in liver of all but Yp mRNA. Kidney GST expression was at least as high as that in liver for GSTs Ya1, Yc, and Yp, while it was substantially lower but still detectable for GSTs Ya2, Yb2, and GSTms. Several of the liver GST class alpha mRNAs, in particular Ya2, were inducible by pretreatment of rats with phenobarbital or isosafrole. In contrast, dexamethasone preferentially induced Yb1, Yb2, and Ya2, while two other inducers of liver drug metabolism, isoniazid and clofibrate, were less effective with respect to GST induction. GSTms mRNA was induced to a small extent or not at all by the agents tested. Treatment of adult male rats with the anticancer drug cisplatin increased liver expression of GST Yc mRNA and suppressed Ya1 mRNA levels with little or no major effect on several other GST mRNAs. Western blot analysis of liver cytosols prepared from the cisplatin-treated rats revealed corresponding changes in GST Yc and Ya protein levels. Comparable changes in liver GST Ya1 and Yc expression were effected by the cisplatin analogue iproplatin but not by carboplatin or transplatin. This pattern of response to these platinum drugs is comparable to that seen with respect to platinum drug-induced gonadal toxicity and modulation of liver cytochrome P450 expression, suggesting a common mechanistic basis for these diverse effects of platinum anticancer drugs on hepatic enzymes of drug metabolism. Together, these studies demonstrate the utility of oligonucleotide probes for phenotyping liver tissue for the expression of GST enzymes that can contribute to anticancer drug metabolism and resistance. They also raise the possibility of drug-drug interactions involving cisplatin and alkylating agent anticancer drugs that can be metabolized in liver by alpha-class GSTs.


Inhibition of cell attachment by selenite. Brief pre-exposure of HeLa cells to micromolar concentrations of selenite resulted in a dose-dependent decrease in the rate of their subsequent attachment to a solid matrix (tissue culture dish). Similar low concentrations of selenite also inhibited colony formation, but only when the cells were exposed prior to their attaching to the dish, not when they were exposed after attachment. This indicates that inhibition of cell proliferation by selenite requires exposure to higher concentrations for longer periods of time. In contrast, selenate, selenomethionine, selenocystine, and sulfite did not affect cell attachment, even at significantly higher concentrations. Thus, the inhibition of cell attachment is a specific effect of selenite. Selenite also inhibited the attachment of cells to bacteriological dishes coated with fibronectin, laminin, or collagen, proteins that are components of the extracellular matrix. There was no inhibition when the tissue culture dishes or the protein-coated dishes were pre-exposed to selenite. There was also no inhibition when the cells were exposed to selenite during the attachment process. Thus, pre-exposure of the cells to selenite was necessary for inhibition of attachment. Since cell attachment has been shown to be an important early step in tumor cell invasion and metastasis, these results suggest a novel mechanism of the anticarcinogenic effect of selenite: inhibition of the attachment of tumor cells to the extracellular matrix.


Expression level of the nm23 gene in clonal populations of metastatic murine and human neoplasms. The purpose of this study was to determine whether nm23 steady-state mRNA expression levels correlate with metastatic potential of mouse K-1735 melanoma cells, human KM12 colon cancer cells, and human SN12 renal cancer cells. Since neoplasms are heterogeneous and contain subpopulations of cells with different metastatic potentials, we analyzed multiple sets of nonmetastatic and metastatic clones isolated from each neoplasm. In addition, we also examined nine somatic cell hybrids produced by the fusion of nonmetastatic and metastatic K-1735 clones. In the mouse melanoma, we found heterogeneity in nm23-1 steady-state expression levels among the clones and hybrids that did not correlate with their metastatic phenotype. Clones isolated from human colon or renal carcinomas expressed similar levels of nm23-HI regardless of metastatic potential in nude mice. All of the human tumor cells were heterozygous for the nm23-HI-specific allelic DNA fragments, with no allelic deletions or gross alterations detected. Since the failure of tumor cells to produce metastasis can be due to multiple deficiencies, these data stress the importance of using independent clones with different metastatic potentials for the analysis of gene regulation of this process.


Fine-scale deletion mapping of the distal long arm of chromosome 6 in 70 human ovarian cancers. To define a small region on chromosome 6q containing a putative tumor suppressor gene for ovarian cancer, we examined loss of heterozygosity in 70 ovarian tumors of three histological types with nine restriction fragment length polymorphism markers located at 6q24-27. Among 33 cancers of serous type that were informative at one or more loci, 17 showed allelic loss at a few or all loci examined, whereas only 1 of 15 mucinous-type tumors and 2 of 12 clear-cell tumors revealed loss of heterozygosity. This result supported our earlier suggestion that alteration of a gene on chromosome 6q may play an important role during development of serous ovarian tumors (Sato et al., Cancer Res., 51: 5118-5122, 1991). Frequent losses were observed between loci defined by CI6-119 (D6S195) at 6q26 and CI6-49 (D6S161) at 6q27. A detailed deletion map indicated a commonly deleted region between loci defined by CI6-111 (D6S193) and CI6-24 (D6S149); these two markers are estimated to be 1.9 cM apart on the basis of linkage analysis. Our results further define a region containing a tumor suppressor gene involved in ovarian carcinoma within an approximately 2-megabase-long segment of chromosome 6q.


Effective alpha-particle-mediated radioimmunotherapy of murine leukemia. The specificity, toxicity, and efficacy of alpha-particle-mediated radioimmunotherapy of murine erythroleukemia was assessed by use of tumor-specific monoclonal antibody 103A labeled with 212Bi. Forty % of the injected dose/g tissue targeted to neoplastic spleens within 1 h after i.v. injection. When 212Bi-103A was injected on day 13 of disease, a dose-dependent response was achieved, as measured by a reduction in splenomegaly and absence of liver metastasis. Mice treated with 212Bi-103A on day 8 of disease showed no histological evidence of erythroleukemia on day 22 and survived significantly longer (median, 118 days) than mice treated with 212Bi-control IgG (78 days) or untreated mice (63 days), indicating successful specific radioimmunotherapy.


Isolation of a prostate carcinoma cell proliferation-inhibiting factor from human seminal plasma and its similarity to transforming growth factor beta. Human seminal plasma (SP) has been known to contain both growth-inhibitory and -stimulatory factors. We attempted to identify a factor that inhibited DNA synthesis in some metastatic prostate cancer cell lines. The SP factor was sensitive to digestion by trypsin, but its activity increased after boiling or dialysis against 1 M acetic acid, by 3- to 4-fold. The SP factor was partially purified using a cation-exchange resin. Apparent molecular mass determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed it to be a M(r) 25,000 protein, and M(r) 13,000 after reduction. This protein strongly inhibited DNA synthesis in two metastatic androgen-independent human prostatic carcinoma cell lines (PC3 and DU145) and the Dunning R3327G rat prostatic adenocarcinoma. It was ineffective on androgen-dependent LNCaP cells. The proliferation-inhibiting activity of this SP protein was specifically and completely abolished by a neutralizing anti-transforming growth factor beta (TGF-beta) antiserum. Furthermore, immunoblot analysis using the anti-TGF-beta antiserum showed the similarity of this protein to TGF-beta. The maximum concentration of this protein in SP was 165 +/- 11.7 ng/ml (mean +/- SD), of which only one-fourth may be present in active form under normal conditions. Identification of a TGF-beta-like protein in SP might also explain the variety of growth and immune modulation properties of human SP.


Structure and expression of human Fli-1 gene. Three ets family members, v-ets, spleen focus forming virus proviral integration 1/Pu.1, and Friend leukemia integration 1 (Fli-1), were shown to be involved in retroviral mediated acute leukemias suggesting that ets family members play a crucial role in transformation. Mouse Fli-1 was shown to be involved in 75% erythroleukemias induced by Friend murine leukemia virus suggesting the possibility that Fli-1 may play a critical role in cellular transformation. Since Fli-1 maps to the mouse chromosome region syntenic with human chromosome 11q23-24, it is tempting to speculate that human Fli-1 may be involved in human sarcomas, leukemias, and lymphomas involving human chromosome 11q23-24. We have isolated complementary DNA clones representing the human homologue of Fli-1 gene. Nucleotide sequence analysis revealed that the human Fli-1 gene codes for a 452-residue protein the predicted amino sequence of which shows 80% homology to the human erg-2 protein previously described. A 3.5-kilobase transcript of the human Fli-1 gene was observed in different cells. Sequence analysis revealed two domains of ets homology, one at the 5' and the other at the 3' end of the Fli-1 gene. This 3'-ets homology domain, which is mainly responsible for DNA binding activity, is seen in all the ets family members; however, the 5'-ets homology region is conserved in only five genes, Fli-1, c-ets-1, ets-2, GABP-alpha, and erg, suggesting a common biological function which is shared among these genes. Interestingly, mouse and human Fli-1 transcripts contain highly homologous 5'-untranslated region suggesting that this conserved region may play an important role in the posttranscriptional regulation of the Fli-1 transcript.


Pharmacokinetic analysis of the perivascular distribution of bifunctional antibodies and haptens: comparison with experimental data. A mathematical model is developed to describe the concentration profiles around individual tumor blood vessels for two-step approaches to cancer treatment. The model incorporates plasma pharmacokinetics, interstitial diffusion, reversible binding between antibody and hapten and between antibody and tumor-associated antigens, and physiological parameters to evaluate present experimental approaches and to suggest new guidelines for the effective use of two-step approaches. Results show considerable interaction between the binding kinetics, initial drug doses, and antigen density, with optimal parameter ranges depending on the desired goal: treatment or detection. The hapten concentration in tumors was found to be nonuniform because of specific binding to antibodies. While binding of the hapten to the bifunctional antibody is necessary for improved retention, too large a binding affinity may lead to very poor penetration of the hapten into regions far away from blood vessels. The time delay between antibody and hapten injection was found to be an important parameter. Longer time delays were found to be advantageous, subject to constraints such as internalization of the antibody and tumor growth during treatment. A proper combination of initial doses for the two species was also seen to be crucial for maximum effectiveness. Comparison of the model with the experimental data of Le Doussal et al. (Cancer Res., 51: 6650-6655, 1991) and Stickney et al. (Cancer Res., 50: 3445-3452, 1990) suggests two novel, yet testable, hypotheses: (a) the early pharmacokinetics of low molecular weight agents can have an important effect on later concentrations using two-step approaches; and (b) metabolism may play an important role in reducing concentrations in the tumor and tumor:plasma concentration ratios. These results should help in the effective design of two-step strategies.


Tumor cell surface-associated binding site for the M(r) 72,000 type IV collagenase. We have studied the capacity of two human breast adenocarcinoma cells, MDA-MB231 and MCF-7, to bind exogenous M(r) 72,000 type IV collagenase by both morphological and radioreceptor binding assays. By indirect immunofluorescence, staining with a specific anti-M(r) 72,000 type IV collagenase antibody was strongly induced when cells were preincubated with the purified enzyme. Scatchard plot analysis indicated the existence of a binding site for the M(r) 72,000 type IV collagenase with high affinity for both cell lines (Kd = 2 x 10(-9) M). These results are the first demonstration of the existence of a tumor cell membrane-associated putative receptor for a member of the matrix metalloproteinase family, as previously evidenced for the urokinase-type plasminogen activator.


Distribution of cells expressing myc proteins in human colorectal epithelium, polyps, and malignant tumors. The myc gene family encodes nuclear phosphoproteins that are thought to play a role in the control of cellular proliferation and differentiation. We have undertaken an immunohistochemical study assessing the expression of myc gene family proteins in individual cells of normal colonic mucosa, colorectal polyps, and colorectal adenocarcinomas. We screened a panel of mouse monoclonal antibodies that we raised against recombinant human c-myc and N-myc proteins for recognition of myc proteins in paraffin tissue sections. Two of these antibodies, H120C69 and H8C150, were selected for indirect immunoperoxidase staining of tissue sections from 16 normal mucosas, 24 polyps, and 30 adenocarcinomas. In normal colon, about 25% of the cells in the lower one-third of the crypts of Lieberkühn stain for myc-related protein. This distribution resembles that of proliferating cells in the crypt. Benign hyperplastic polyps resemble normal mucosa in their myc staining pattern, with about 25% of the cells positive. In adenomatous polyps, the putative precursors of adenocarcinomas, from 50 to 100% of the cells stain positively for myc protein. In these cases, stained cells extend to the luminal surface, consistent with the previously reported expansion of the proliferation zone in these lesions. All adenocarcinomas examined had increased levels of myc protein relative to normal mucosa. The tumor cells exhibited markedly heterogeneous myc staining patterns, both among different tumors and, in some cases, within a single tumor. Comparison with Ki-67 monoclonal antibody staining indicates that myc protein expression in many tumors is uncoupled from cellular proliferation. Surprisingly, we observed increased numbers of myc-expressing cells and increased levels of myc protein in histologically normal colon directly adjacent to tumor, suggesting that many colorectal carcinomas secrete growth factors that activate gene expression in neighboring normal mucosa.