Margaret LaCava

Increased cell growth and tumorigenicity in human prostate LNCaP cells by overexpression to cyclin D1

Deregulated expression of cyclin D1 has been found in several types of human tumors. In order to investigate factors involved in human prostate cancer progression, we studied the effects of cyclin D1 overexpression on human prostate cancer cell proliferation and tumorigenicity by transfecting LNCaP cells with a retroviral vector containing human cyclin D1 cDNA. When compared to the parental and control-vector transfected LNCaP cells, these cyclin D1-transfected cells had more cells in S-phase and lower growth factor requirements. Furthermore, these cells grew more in androgen-free medium. We also detected higher levels of Rb phosphorylation and E2F-1 protein levels in LNCaP/cyclin D1 cells than that in the parental and vector control cells in medium with or without androgen. Cyclin D1 transfected clones formed tumors more rapidly than control and parental cells. These tumors were refractory to the androgen-ablation treatment by castration, whereas tumors from parental and vector-control LNCaP cells regressed within 4 weeks after castration. These results suggest that overexpression of cyclin D1 changes the growth properties, increases tumorigenicity and decreases the requirement for androgen stimulation in LNCaP cells both in vitro and in vivo.

cdk4 deficiency inhibits skin tumor development but does not affect normal keratinocyte proliferation

Most human tumors have mutations that result in deregulation of the cdk4/cyclin-Ink4-Rb pathway. Overexpression of D-type cyclins or cdk4 and inactivation of Ink4 inhibitors are common in human tumors. Conversely, lack of cyclin D1 expression results in significant reduction in mouse skin and mammary tumor development. However, complete elimination of tumor development was not observed in these models, suggesting that other cyclin/cdk complexes play an important role in tumorigenesis. Here we described the effects of cdk4 deficiency on mouse skin proliferation and tumor development. Cdk4 deficiency resulted in a 98% reduction in the number of tumors generated through the two-stage carcinogenesis model. The absence of cdk4 did not affect normal keratinocyte proliferation and both wild-type and cdk4 knockout epidermis are equally affected after topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in epidermal hyperplasia. In similar fashion, cdk4 knockout keratinocytes proliferated well in an in vivo model of wound-induced proliferation. Biochemical studies in mouse epidermis showed that cdk6 activity increased twofold in cdk4-deficient mice compared to wild-type siblings. These results suggest that therapeutic approaches to inhibit cdk4 activity could provide a target to inhibit tumor development with minimal or no effect in normal tissue.

Cyclin D2 Overexpression in Transgenic Mice Induces Thymic and Epidermal Hyperplasia whereas Cyclin D3 Expression Results Only in Epidermal Hyperplasia

In a previous report, we described the effects of cyclin D1 expression in epithelial tissues of transgenic mice. To study the involvement of D-type cyclins (D1, D2, and D3) in epithelial growth and differentiation and their putative role as oncogenes in skin, transgenic mice were developed which carry cyclin D2 or D3 genes driven by a keratin 5 promoter. As expected, both transgenic lines showed expression of these proteins in most of the squamous tissues analyzed. Epidermal proliferation increased in transgenic animals and basal cell hyperplasia was observed. All of the animals also had a minor thickening of the epidermis. The pattern of expression of keratin 1 and keratin 5 indicated that epidermal differentiation was not affected. Transgenic K5D2 mice developed mild thymic hyperplasia that reversed at 4 months of age. On the other hand, high expression of cyclin D3 in the thymus did not produce hyperplasia. This model provides in vivo evidence of the action of cyclin D2 and cyclin D3 as mediators of proliferation in squamous epithelial cells. A direct comparison among the three D-type cyclin transgenic mice suggests that cyclin D1 and cyclin D2 have similar roles in epithelial thymus cells. However, overexpression of each D-type cyclin produces a distinct phenotype in thymic epithelial cells.

Definitive functional evidence for a tumor suppressor gene on human chromosome 7q31.1 neighboring the Fra7G site.

We have previously shown that loss of heterozygosity (LOH) on human chromosome (hchr) 7 at q31.1 is common in a variety of tumors of epithelial origin. Frequent LOH of a specific chromosomal marker is indicative of a closely linked tumor suppressor gene (TSG). However, recent reports have also indicated that such a high frequency of LOH could be due to the presence in this region of the second most common aphidicolin-inducible fragile site in the human genome (Fra7G). To address this controversy, we introduced single copies of hchr7 or hchr12 into a highly aggressive human prostate carcinoma cell line (PC3) by microcell-mediated transfer. The tumorigenicity of six clones of PC3/hchr7 hybrids and three clones of PCRhchr12 hybrids, obtained in four separate fusion experiments, were studied in BALB/c nude mice. All but one of the PC3/hchr7 hybrids increased tumor latency by at least twofold, whereas none of the PC3/hchr12 hybrids delayed tumor onset. No differences in the in vitro growth rate were observed among any of the cell lines assayed (parental and hybrids) suggesting that the observed tumor suppression was due to factors other than cell cycle regulation. Deletion mapping of the PC3/hchr7 tumors obtained after reversion to the malignant phenotype revealed a common region of loss centred around 7q31.1, supporting the TSG hypothesis. The smallest commonly deleted region was ∼1.5 Mb in size and flanked by the markers D7S486 and D7S655.

Deregulated expression of cell-cycle proteins during premalignant progression in SENCAR mouse skin

It is now evident that several genes encoding regulatory activities that control the mammalian cell cycle, particularly some that control the progression of quiescent cells through G1 and into S phase, are targets for alterations that underlie the development of neoplasms. Here, we made a sequential study of alterations in cell cycle protein expression and complex formation among cyclin, cyclin dependent kinases (CDKs) and CDK inhibitors (CKIs) during premalignant progression in SENCAR mouse skin tumors. Changes in the level of expression were observed in positive (cyclin D1, D2, and E2F family members) and negative regulators (p16Ink4a, p57Kip2) of the cell cycle. Also, we observed the formation of cyclin/CDK/CKI complexes. The amounts of these proteins and complexes increased substantially at specific times during promotion but not during malignant conversion to carcinomas. These data show that deregulation of growth control occurs in benign tumors and that subsequent mutations not involved cell-cycle regulation are probably necessary to induce invasive behavior.

Transgenic expression of cyclin-dependent kinase 4 results in epidermal hyperplasia, hypertrophy, and severe dermal fibrosis.

In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the D-type cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27(Kip1) to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Also, abnormal expression of keratin 6 associated with the hyperproliferative phenotype was observed in transgenic epidermis. This model provides in vivo evidence for the role of CDK4 as a mediator of proliferation in epithelial cells independent of D-type cyclin expression.

Increased expression of mutated Ha‐ras during premalignant progression in SENCAR mouse skin

The ras proto‐oncogene family products are membrane‐associated, guanine nucleotide–binding proteins that serve as a molecular switch for signal transduction pathways in a diverse array of organisms. In the mouse skin two‐stage carcinogenesis model, a specific point mutation in Ha‐ras codon 61 is responsible for the initiation event. Here we investigated whether Ha‐ras protein and mRNA expression change during premalignant progression. Also, we assessed the Ha‐ras mutated allele after these changes. To those ends, we analysed the Ha‐ras expression profiles in normal and hyperplastic skin, papillomas, and squamous cell carcinomas by western blotting, reverse transcription–polymerase chain reaction, and in situ hybridization. Increased levels of Ha‐ras expression were observed at specific times during promotion. These changes were followed by an increase in the level of expression of the Ha‐ras mutated allele. These results suggest that increased expression of Ha‐ras mutated alleles may have an important role during premalignant progression. Mol. Carcinog. 26:150–156, 1999.

Genetic analyses of mouse skin tumor progression susceptibility using SENCAR inbred derived strains.

Susceptibility to tumor development varies among individuals in the human population. This variability can also be found among different strains of mice, particularly in the mouse skin chemical carcinogenesis model. The genetic mechanisms underlying mouse skin tumor susceptibility are not fully understood. The SENCAR stock has been found to be the most sensitive mice for skin carcinogenesis studies; however, little is known about the genes underlying tumor susceptibility, particularly, those involved in tumor progression. Experiments with the SSIN/Sprd mice, an inbred strain derived from the outbred SENCAR stock, suggested that papilloma development, tumor promotion, and their conversion into squamous cell carcinomas (SCCs), progression, are regulated by different genes. In the highly sensitive SSIN/Sprd mice, papillomas rarely progress to SCC. Using crosses between the outbred SENCAR and the SSIN/Sprd mice, we previously determined that papilloma progression in the SENCAR stock could be controlled by at least one autosomal dominant gene. However, the outbred nature of the SENCAR stock precluded us from extending those findings. More recently, another inbred strain was developed from the outbred SENCAR stock, the SENCARB/Pt. These mice have similar tumor promotion sensitivity to the SSIN/Sprd but in contrast, have high papilloma progression susceptibility, similar to the outbred original stock. In the present study, we generated F1, F2, and backcross hybrids between the SSIN/Sprd and SENCARB/Pt mice to determine a possible model for tumor progression susceptibility and to map the putative tumor susceptibility genes. Our tumor data suggests that papilloma progression susceptibility in the SENCAR mouse skin model could be genetically determined by one susceptibility gene. Our preliminary linkage analysis failed to identify one strong susceptibility locus to confirm this but provided some evidence for at least one possible susceptibility locus in mouse chromosome 14.