Renato Dulbecco

The properties of a mammary gland cancer stem cell

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing a single cancer-initiating cell with stem cell properties has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell derived from rat mammary adenocarcinoma has the following properties: the differentiation potential to generate all of the cell lineages of the mammary gland; the ability to generate branched duct-like structures that recapitulate morphologically and functionally the ductal–alveolar-like architecture of the mammary tree; and the capacity to initiate heterogeneous tumors in nonobese diabetic-SCID mice. In addition, we show that cultured cells derived from tumors generated by a single LA7 cell-injection have properties similar to LA7 cells, can generate all of the cell lineages of the mammary gland, and recapitulate the ductal–alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multilineage differentiation potential, and single-cell tumor-initiation potential suggest that LA7 cells are cancer stem cells and can be used as a model system to study the dynamics of tumor formation at the single-cell level.

Gene expression profiles of epithelial cells microscopically isolated from a breast-invasive ductal carcinoma and a nodal metastasis.

Expression profiles of breast carcinomas are difficult to interpret when they are obtained from tissue in toto, which may contain a large proportion of non-cancer cells. To avoid this problem, we microscopically isolated cells from a primary invasive ductal carcinoma of the breast and from an axillary node harboring a metastatic breast carcinoma, to obtain pure populations of carcinoma cells (≈500) and used them for serial analysis of gene expression. The expression profiles generated from both populations of cells were compared with the profile of a disease-free mammary epithelium. We showed that the expression profiles obtained are exclusive of carcinoma cells with no contribution of non-epithelial cells. From a total of 16,939 unique tags analyzed, we detected 559 statistically significant changes in gene expression; some of these genes have not been previously associated with breast cancer. We observed that many of the down-regulated genes are the same in both cancers, whereas the up-regulated genes are completely different, suggesting that the down-regulation of a set of genes may be the basic mechanism of cancer formation, while the up-regulation may characterize and possibly control the state of evolution of individual cancers. The results obtained may help in characterizing the neoplastic process of breast cancer.

Isolation and characterization of temperature-sensitive mutants of simian virus 40.

Abstract Six temperature-sensitive (ts) mutants of Simian Virus 40 (SV40) which produce plaques at 33 but not at 40 C were isolated using either nitrosoguanidine or hydroxylamine as mutagens. One cycle of growth of the mutants at 40 C after infection with viral DNA was restricted, showing that the block to multiplication at 40 C is not due to failure in adsorption, penetration or uncoating. The mutants are more heat labile than wild-type virus, and are temperature-independent for synthesis of infectious viral DNA and for transformation. Four of the six mutants could be assigned to two complementation groups, and probably identify two genes for structural virion proteins, whose functions are not required for transformation.

A temperature-sensitive mutant of simian virus 40 affecting transforming ability

Abstract In productive infection of monkey BSC-1 cells, ts 640, a temperature-sensitive (ts) mutant of simian virus 40 (SV40), was defective in its ability to synthesize infectious viral DNA and infectious viral particles at the restrictive temperature of 40 °, but not at the permissive temperature of 33 °. Growth of the mutant after infection with viral DNA was also temperature-sensitive, indicating that the block to multiplication at 40 ° is not due to failure in adsorption, penetration or uncoating. Results of mixed infections with other ts mutants showed that the gene identified in this mutant is different from those two cistrons which had been suggested to specify virion proteins. In nonproductive infection of rat 3Y1 cells, ts 640 was temperature-sensitive in its transforming ability at 40 ° but not at 33 °, providing evidence that cell transformation by SV40 is under the control of a viral gene function. The transformed cells obtained at 33 ° appeared not to revert to normal when shifted to 40 °, as judged from their colonial morphology under the standard culture conditions.

Induction of cellular DNA synthesis by polyoma virus. II. Increase in the rate of enzyme synthesis after infection with polyoma virus in mouse kidney cells

Abstract An induction of cellular DNA synthesis and an increase in the activity of three enzymes involved in DNA synthesis, dCMP deaminase, TdR Kinase, and DNA polymerase, following the infection of mouse kidney cells with polyoma virus was described previously (Dulbecco et al. , 1965). Experiments described in this paper elucidate the mechanism of the increase in enzymatic activities of dCMP deaminase and TdR kinase. Three alternative mechanisms are considered: (1) an increase in the rate of enzyme synthesis, (2) a stabilization of labile enzyme molecules, and (3) an activation of enzyme activity. Experimental evidence is presented which rules out the latter two possibilities. We conclude therefore that the rate of synthesis of dCMP deaminase and TdR kinase is increased as a result of polyoma infection.

Polyoma virus proteins: I. Multiple virion components

Abstract The polypeptide components of highly purified polyoma virions were separated by electrophoresis on SDS-polyacrylamide gels. Seven distinct virion polypeptides were reproducibly observed, and at least six of these components are unique polypeptides, not aggregates. The sum of the molecular weights estimated for these virion polypeptides nearly equals or exceeds the coding capacity of the viral DNA. The major capsid protein, P2, contains 50–70% of the radioactivity and has an estimated molecular weight of 48,000 daltons. Three small (MW 15,000–19,000) polypeptides, which have a high lysine to valine ratio, are apparently “internal” proteins with an affinity for viral DNA. Several possible explanations for the large number of minor virion polypeptides are discussed.

From the molecular biology of oncogenic DNA viruses to cancer

Oncogenic viruses, able to elicit tumour formation in animals, have been on the scientific scene for many years. After the early discovery of Ellerman and Bang at the beginning of this century, Peyton Rous opened up the field in its second decade and in prophetic words gave a good hint of things to come. However, these discoveries were soon forgotten and only after a long eclipse was interest in oncogenic viruses revived in the fifties. My involvement in this field began at that time when Rubin and Temin worked in my laboratory with the Rous Sarcoma Virus. When in 1958 polyoma virus, a new oncogenic virus with different properties, was isolated, I jumped at the new opportunity and started working with it. Within a short time polyoma virus became the main interest of my laboratory, to be joined, a few years later by SV40, another papovavirus. It became clear fairly soon that the molecular biology of these viruses could be worked out, and I set out to find the molecular basis of cancer induction. The results that I and a number of brilliant young collaborators have obtained during the following fifteen years have brought us close to that goal. I will review the most interesting steps of our work and will then ask some questions concerning the nature of cancer and about perspectives for prevention and treatment. I stress the relevance of my work for cancer research because I believe that science must be useful to man.

Abortive Transformation by the Tsa Mutant of Polyoma Virus

The Tsa mutant of polyoma virus (PyTsa) is a conditional lethal mutant isolated by Fried1. PyTsa grows normally at the permissive temperature (31.5° C), but at the nonpermissive temperature (38.5° C) there is a failure of normal viral DNA synthesis, and of vegetative growth in mouse cells. Transformation of hamster cells by PyTsa is also inhibited at 38.5° C; this raises the question, Is the mutant gene required simply for the viral induced change in the cell physiology or is it concerned with the stable propagation of the transformed state? These alternatives can be distinguished by the phenomenon of abortive transformation2.

Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland

The study of the development of the mammary gland at the molecular level in the animal is difficult because of the complex tissue organization of the gland. We have previously developed an in vitro system for genetic analysis of mammary cell differentiation, based on the cell line LA7 clonally derived from a rat mammary adenocarcinoma. This cell line, after induction with DMSO, differentiates forming structures called domes. This process is under strict gene regulation, and we have previously identified several of the genes involved. In the present paper, we have defined the meaning of dome formation in relation to mammary development, by showing that treatment of LA7 cells with the lactogenic hormones hydrocortisone and prolactin induces dome formation; in the animal, these hormones precede and accompany milk production. Moreover, dome formation is accompanied by expression within the cells of the milk protein genes WDMN1 and β-casein, which are differentiation markers for the gland during pregnancy and lactation. We also show that two proteins, highly expressed in the mammary gland during lactation, HSP90-β and annexin I, are strongly expressed in DMSO-induced LA7 cells. Both proteins are essential in the formation of domes because when their synthesis is blocked by antisense RNA oligonucleotides, dome formation is abolished. Thus our in vitro system is a model for lobulo-alveolar development, and the genes identified in the pathway of dome formation are likely to be involved in the early differentiation steps occurring in the rat mammary gland during pregnancy and lactation.

Proteomic dissection of dome formation in a mammary cell line: Role of tropomyosin-5b and maspin

In this work we extended the study of genes controlling the formation of specific differentiation structures called “domes” formed by the rat mammary adenocarcinoma cell line LA7 under the influence of DMSO. We have reported previously that an interferon-inducible gene, rat-8, and the β-subunit of the epithelial sodium channel (ENaC) play a fundamental role in this process. Now, we used a proteomic approach to identify proteins differentially expressed either in DMSO-induced LA7 or in 106A10 cells. Two differentially expressed proteins were investigated. The first, tropomyosin-5b, strongly expressed in DMSO-induced LA7 cells, is needed for dome formation because its synthesis inhibition by the antisense RNA technology abolished domes. The second protein, maspin, strongly expressed in the uninduced 106A10 cell line, inhibits dome formation because 106A10 cells, transfected with rat8 cDNA (the function of which is required for the organization of these structures), acquired the ability to develop domes when cultured in presence of an antimaspin antibody. Dome formation in these cultures are accompanied by ENaC β-subunit expression in the absence of DMSO. Therefore, dome formation requires the expression of tropomyosin-5b, in addition to the ENaC β-subunit and the rat8 proteins, and is under the negative control of maspin.