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Epidermal growth factor (EGF) markedly stimulates cell proliferation in a variety of mammalian systems. For this reason, EGF and factors related to EGF were examined for a possible role in the promotion and maintenance of the uncontrolled growth state that is a characteristic of malignant neoplasias. Phorbol ester tumor promoters, compounds that are capable of promoting tumors in the mouse skin carcinogenesis assay, act synergistically with EGF to stimulate DNA synthesis in cultured fibroblasts despite an inhibitory effect on the binding of EGF to its cellular receptor. Sparing of EGF degradation in combination with recovery of EGF binding was postulated to be responsible for the increased role of DNA synthesis in cells exposed to the phorbol esters. The hypothesis is presented that the hyperplasiogenic component of tumor promotion may be mediated, at least in part, by local alterations in growth factor levels. Recent evidence suggests the existance of a family of molecules related to EGF as defined by the ability to bind to the EGF receptor. These factors (transforming growth factors) appear to be responsible for the phenotypic alterations characteristic of transformed cells. Using a radioreceptor assay, two EGF-like factors were isolated from mouse submaxillary gland. These factors were not transforming growth factors and appeared to be modified EGF molecules. Two EGF-like factors, molecular weight 27,000 and 13,000, were identified in medium conditioned by Rous sarcoma virus (RSV)-transformed cells and were shown to possess the characteristics of a transforming growth factor. In addition, rat fetus extracts contained a 55,000 molecular weight EGF-like factor with the properties of a transforming growth factor. The EGF-effector system may therefore play an important role in embryonic development and in the maintenance of the neoplastic phenotype. The difference in the molecular weights of the RSV-factor and the fetus factor indicates that there are numerous members of the class of EGF-like molecules, and that RSV-transformation probably does not induce the re-expression of a fetal growth stimulatory factor. The results of these experiments suggest that EGF and EGF-like factors are biologically important growth-stimulating molecules that must be tightly regulated to maintain normal physiological conditions.

This study elucidates the temporal expression and regulation of transglutaminase (TGase) and ornithine decarboxylase (ODCase) during cell proliferation and differentiation. In synchronized CHO cells, there were two peaks of TGase activity expressed in G₁ and a smaller peak of activity in mid S phase. ODCase exhibited a single peak of expression in mid G₁ which was inhibited by the administration of both cycloheximide and actinomycin D. In contrast, the increase in TGase activity was not inhibited at any time measured by administration of either cycloheximide or actinomycin D to these cells. TGase activity in CHO cells was not affected by the addition of analogs of cyclic AMP, whereas ODCase activity was increased at all times measured. Retinol administration increased TGase activity 1 hr after release in CHO cells and the activity remained elevated for 4 hr. Retinol administration resulted in the inhibition of ODCase expression in these cells. The administration of α-melanocyte-stimulating hormone (MSH) to mouse melanoma cells resulted in a biphasic increase of TGase activity and a single peak of ODCase activity within 7 hr. In melanoma cells, addition of cycloheximide abolished the first peak of TGase activity but not the second peak. Actinomycin D did not inhibit either peak of TGase expression. The administration of both cycloheximide and actinomycin D inhibited ODCase activity after MSH stimulation. Analogs of cyclic AMP, when added to log phase mouse melanoma cells, increased ODCase but not TGase activity at all points measured. In these cells, retinoic acid plus MSH markedly enhanced the activity of the initial TGase peak compared to MSH alone. ODCase expression was attenuated with retinoic acid plus MSH. Dexamethasone (DEX) induced the first peak of TGase activity but not the second peak seen with MSH administration alone. Administration of DEX resulted in a peak expression of ODCase activity approximately 30% of that seen with MSH alone. In general, chelation of extracellular calcium with EGTA totally blocked ODCase expression with MSH, retinoic acid or DEX. Partial or total ablation of TGase expression was seen with addition of MSH or retinoic acid, but very little inhibition of this enzyme was evident when EGTA was added with DEX or DEX plus MSH. Addition of calcium after all CA⁺⁺-blocks restored the expression of both enzymes.

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Aberrant cell proliferation is a fundamental feature of cancer. It is thus not unexpected that many studies have been devoted to the exploration of tumour cell proliferation as a potential indicator of outcome. Indeed, in most malignancies, high expression of proliferation markers and more accurately, increased expression of proliferation-related genes have been strongly associated with poor outcome. In colorectal cancer (CRC), however, discordant results have been reported and the prognostic significance of cell proliferation has not been demonstrated in this type of cancer. As these results were mostly based on the subjective assessment of a single proliferation marker, this work set out to evaluate the association between the proliferative activity of CRCs and their malignant potential using an objective microarray-based multi-gene proliferation signature. In the first step, a gene proliferation signature (GPS) was derived from analysis of a CRC cell line model. Ten CRC cell lines were harvested under semi- and fully-confluent conditions to obtain RNA from two stages that differed in their proliferative activity. Gene expression profiles of the two growth stages were analyzed on oligonucleotide arrays and a GPS was identified by gene ontology analysis of differentially expressed genes. In the second step, the performance of the signature to classify patients into prognostic groups was examined using two independent cohorts of primary CRC tumours (cohort A: 73 tumours in stages I-IV, cohort B: 55 tumours in stages I-II). Further, the signature was applied to a population of liver metastases to establish its association with the malignant potential of CRC. Finally, the capacity of the signature to detect clinically distinct CRC populations was compared with that of the proliferation marker Ki-67 in a classic immunohistochemical approach. The GPS consisted of 38 mitotic cell cycle genes, which were over-expressed in actively cycling cells relative to growth-inhibited cells in vitro. Intriguingly, a reduced GPS expression was associated with (i) the presence of lymph node or distant metastasis in cohort A, (ii) an increased risk of recurrence, and (iii) shorter overall and recurrence-free survival in both cohorts of primary CRCs (p<0.05). While the association between the GPS and clinical outcome was not independent of the disease stage in cohort A, reduced GPS expression was an independent predictor of outcome in cohort B. Importantly, adjuvant therapy had no impact on this association. Furthermore, GPS expression was reduced in CRC liver metastases, confirming that decreased proliferation is an indicator of the malignant potential in CRC. While reduced proliferation in liver metastases was also observed by Ki-67 immunostaining, the classic proliferation marker was not able to stratify primary CRCs into different prognostic groups. To the best of our knowledge, this study is the first to report an association between the reduced expression of a multi-gene proliferation signature and poor outcome in cancer. This finding contradicts the long-held belief that increased proliferation is an indicator of tumour malignancy. In contrast to many other cancers, reduced proliferation appears to be a significant component of a biological signature associated with malignant potential of CRC tumours. Investigating the reasons why CRC differs from other cancer types may provide insights into important underlying biological mechanisms. If this association can be verified in larger cohorts, the GPS may have important clinical implication for identification of high-risk early stage CRC patients.

Includes summary / Includes bibliography / [586] leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Dept. of Anatomy and Histology, 1988