Thomas H. Norwood

Molecular Targeting of Platelet-Derived Growth Factor B by Imatinib Mesylate in a Patient With Metastatic Dermatofibrosarcoma Protuberans

PURPOSE Dermatofibrosarcoma protuberans is caused by activation of the platelet-derived growth factor B (PDGFB) receptor, a transmembrane tyrosine kinase. We investigated the response of dermatofibrosarcoma protuberans to the tyrosine kinase inhibitor imatinib mesylate. PATIENTS AND METHODS A patient with unresectable, metastatic dermatofibrosarcoma protuberans received imatinib mesylate (400 mg bid). Response to therapy was assessed by [18F]fluorodeoxyglucose (FDG) positron emission tomography, magnetic resonance imaging, and histopathologic and immunohistochemical evaluation. RESULTS The patient was treated for 4 months with imatinib mesylate. The hypermetabolic uptake of FDG fell to background levels within 2 weeks of treatment, and the tumor volume shrank by over 75% during the 4 months of therapy, allowing for resection of the mass. There was no residual viable tumor in the resected specimen, indicating a complete histologic response to treatment with imatinib mesylate. CONCLUSION Imatinib mesylate is highly active in dermatofibrosarcoma protuberans. The dramatic response seen in this patient demonstrates that inhibition of PDGFB receptor tyrosine kinase activity can significantly impact viability of at least one type of solid tumor.

Dimethyl sulfoxide enhances polyethylene glycol-mediated somatic cell fusion

The efficiency of fusion of human diploid cells by polyethylene glycol was greatly enhanced by addition of dimethyl sulfoxide. The extent of fusion was directly proportional to the concentrations of both of these compounds. At all except the highest concentrations, cell loss was moderate to minimal and perturbation of cell cycle function as measured by [3H]thymidine labeling indices and mitotic indices was minimal in the surviving cells. This technique is potentially useful for heterokaryon studies as well as for the isolation of hybrids of mammalian somatic cells.

Mutation at the Polymerase Active Site of Mouse DNA Polymerase δ Increases Genomic Instability and Accelerates Tumorigenesis

ABSTRACT Mammalian DNA polymerase δ (Pol δ) is believed to replicate a large portion of the genome and to synthesize DNA in DNA repair and genetic recombination pathways. The effects of mutation in the polymerase domain of this essential enzyme are unknown. Here, we generated mice harboring an L604G or L604K substitution in highly conserved motif A in the polymerase active site of Pol δ. Homozygous Pold1L604G/L604G and Pold1L604K/L604K mice died in utero. However, heterozygous animals were viable and displayed no overall increase in disease incidence, indicative of efficient compensation for the defective mutant polymerase. The life spans of wild-type and heterozygous Pold1+/L604G mice did not differ, while that of Pold1+/L604K mice was reduced by 18%. Cultured embryonic fibroblasts from the heterozygous strains exhibited comparable increases in both spontaneous mutation rate and chromosome aberrations. We observed no significant increase in cancer incidence; however, Pold1+/L604K mice bearing histologically diagnosed tumors died at a younger median age than wild-type mice. Our results indicate that heterozygous mutation at L604 in the polymerase active site of DNA polymerase δ reduces life span, increases genomic instability, and accelerates tumorigenesis in an allele-specific manner, novel findings that have implications for human cancer.

Comparative heterokaryon study of cellular senescence and the serum-deprived state

Abstract Autoradiographic patterns of DNA replication in serum-deprived human diploid fibroblast-like cells (HDFC) and “senescent” HDFC have been compared in two types of heterokaryons. Each was fused to low passage, proliferating HDFC and, in separate experiments, to HeLa cells. Sequential 1 h pulses with [3H]thymidine were initiated at short intervals following fusion. In all hybridizations serum-deprived and senescent cells behaved identically. Upon fusion to HeLa cells, DNA synthesis in the quiescent nuclei occurred in a wave between 3 and 30 h after fusion. When either serum-deprived or senescent HDFC were fused to young proliferating HDFC, the nuclei of the latter were blocked from entering the S phase if fusion occurred at least 3 h before the G/S boundary. These findings are consistent with the interpretation that one or more crucial steps in G0 occurs 3 h before the G1/S interface. That young serum-deprived (G0) HDFC behave identically to senescent cells in these hybridization studies suggests that the mechanism of arrest in each state might share a final common pathway, and a model based on these observations is proposed.

Do Hyperplastoid Cell Lines “Differentiate Themselves to Death”?

Hayflick and Moorhead (1) clearly differentiated between two classes of mammalian cell lines: 1) Those typified by HeLa are apparently immortal and may serve as models for the study of neoplastic cell proliferation; we refer to them as “neoplastoid” 2) Those typified by WI-38 and by human skin fibroblast cultures eventually cease replicating and may be useful as models for the study of hyperplastic cellular proliferation or wound healing; consequently, we refer to them as “hyperplastoid.” Martin and Sprague (2) have recently tabulated some 21 parameters which have been claimed to differentiate between these two classes of cell lines. In mass cultures, the replicative life-span is currently among the most unambiguous differential parameters. Individual clones of either type of culture may cease proliferating, however, and it is this phenomenon which we refer to as “clonal senescence.” In the case of human diploid somatic cells, it is probable that some thousands of such clones have been followed in many different laboratories and to the best of our knowledge, all of them eventually stop growing, unless they are induced to undergo malignant transformation. Curiously, much less is known about the replicative life histories of individual clones and sub-clones of neoplastoid cells, even though they are comparatively easy to clone.

A neuroendocrine/small cell prostate carcinoma xenograft-LuCaP 49.

The late stages of progression of prostate carcinoma are typically characterized by an androgen-insensitive, rapidly proliferative state. Some late-stage tumors are composed predominantly of neuroendocrine cells. Virtually no animal models of a neuroendocrine/small cell variant of prostate carcinoma are available for experimental studies. We report a human neuroendocrine/small cell prostate carcinoma xenograft that was developed from a nodal metastasis of a human prostate carcinoma and that has been propagated as serial subcutaneous implants in severe combined immunodeficient mice for >4 years. Designated LuCaP 49, all tumor passages exhibit a neuroendocrine/small cell carcinoma phenotype-insensitivity to androgen deprivation, expression of neuroendocrine proteins, lack of expression of prostate-specific antigen or androgen receptor, and an unusually rapid growth (a doubling time of 6.5 days) for prostate cancer xenografts. Genetically this tumor exhibits loss of heterozygosity for the short arm of chromosome 8 and has a complex karyotype. This xenograft should prove to be useful in the investigation of mechanisms underlying the androgen-insensitive state of progressive prostate carcinoma.

Inhbition of DNA synthesis in young cycling human diploid fibroblast-like cells upon fusion to enucleate cytoplasts from senescent cells

Previous studies have demonstrated that upon fusion with actively cycling human diploid fibroblast-like (HDFL) cells, senescent HDFL are capable of inhibiting the young nucleus from entry into DNA synthesis (Norwood et al. (1974) [10]; Rabinovitch et al. (1980) [1]). These studies have been interpreted as evidence in favor of the existence of cell cycle inhibitors in senescent cells. Enucleate cytoplasts derived from senescent HDFL cells are also capable of inhibiting the young nucleus from entry into DNA synthesis upon fusion with whole cycling HDFL cells. In contrast, no inhibition was observed in fusions between enucleate young cells and whole cycling cells. These results provide evidence for transmissible cell cycle-inhibitory factors in the cytoplasm of senescent cells.

The relationship between the rate of entry into S phase, concentration of DNA polymerase α, and cell volume in human diploid fibroblast-like monokaryon cells☆

We have examined the kinetic relationship between the rate of entry into the S phase in human diploid fibroblast-like (HDFL) monokaryon cells and (1) the concentration of DNA polymerase alpha activity and (2) the cell volume. In the former studies, a first-order dependence between the rate of entry into the S phase and the concentration of DNA polymerase alpha activity was observed, consistent with the enzyme, or a coregulated factor, being rate limiting for this metabolic process. Examination of the nature of the dependence of the rate of entry into the S phase upon cell volume revealed a more complex relationship. The results obtained in studies with synchronized cultures are consistent with the presence of two to three rate-limiting reactants when cell volume is the independent variable. Studies with asynchronous HDFL cell cultures revealed that the smallest cells in the G1 population, presumably the early G1 cells, enter the S phase at an increasing rate as a function of cell volume up to a certain size, beyond which the cells enter at a decreasing rate similar to that observed in the studies with the synchronized cultures. Similar studies examining the relationship between cell volume and the rate of entry into S phase in three established immortal cell lines revealed positive correlation between the rate of entry into S phase and cell volume throughout the size range of the G1 population. This latter observation suggests that the factors involved in the initiation of the S phase may be present in concentrations that are not rate limiting in immortal cell lines.

Evidence for clonal attenuation of growth potential in hela cells

SummaryThe growth of primary clones and serial subclones of HeLa cells and of diploid human fibroblast-like cells were compared both in the presence and absence of feeder layers; the latter had no significant effects upon the results. Clones and subclones of both cell types displayed great heterogeneity in growth rates, typically with a bimodality of growth distributions. Serial passages of clones selected on the basis of superior rates of proliferation showed attentuation of growth potentials; the extent of such attentuations was much less in the case of HeLa cells, suggesting at least one possible basis for the differences in long-term growth potential between these two classes of cell lines.

Chromosome 7 short arm deletion and craniosynostosis. A 7p-syndrome.

SummaryA patient with craniosynostosis and a small deletion of part of the short arm of chromosome 7 is described. A review of the literature indicates that craniosynostosis has occurred in at least four of the five infants (the fifth having microcephaly) affected by structural changes (resulting in deletion) within the terminal region of the short arm of chromosome 7.