Rosalind J. Jackson

Loss of p21WAF1/CIP1 accelerates Ras oncogenesis in a transgenic/knockout mammary cancer model.

Upregulation of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and subsequent cell growth arrest or senescence is one mechanism by which normal cells are believed to respond to stress induced by the constitutively activated GTPase Ras. We hypothesize that in the absence of p21, the onset of Ras-dependent oncogenesis is accelerated. To test this hypothesis, we crossed MMTV/v-Ha-ras transgenic mice into a p21-deficient background. By 63 days of age, all 8 ras/p21−/− mice developed either malignant (mammary and/or salivary adenocarcinomas) or benign (Harderian hyperplasia) tumors. In contrast, by the same age, only one out of nine of the ras/p21+/+ mice developed a tumor. Furthermore, by 94 days of age, half of the ras/p21−/− mice, but none of the ras/p21+/+ mice, developed mammary tumors. p21-deficiency also accelerated the development of salivary (T50=66 days for ras/p21−/− vs T50=136 days for ras/p21+/+) and Harderian (T50=52 days for ras/p21−/− vs T50>221 days for ras/p21+/+) tumors. Furthermore, two out of the eight ras/p21−/− mice had metastatic lesions, one in its lungs, the other in its abdomen. None of the nine ras/p21+/+ mice had metastatic lesions. By 4 months of age, the mammary tumor multiplicity was 10-fold greater in ras/p21−/− (average 3.40 tumors/mouse) than in ras/p21+/+ (average 0.33 tumor/mouse) mice. However, once the tumors appeared, their growth rate, apoptosis level, and mitotic index were not affected by the loss of p21, suggesting that loss of p21 is critical in early but not late eventsof Ras oncogenesis. Altogether, the results show that tumor onset in MMTV/v-Ha-ras mice is p2l-dependent with loss of p2l associated with earlier tumor appearance and increased tumor multiplicity and aggressiveness.

Loss of the cell cycle inhibitors p21(Cip1) and p27(Kip1) enhances tumorigenesis in knockout mouse models.

Events that contribute to tumor formation include mutations in the ras gene and loss or inactivation of cell cycle inhibitors such as p21Cip1 and p27Kip1. In our previous publication, we showed that mice expressing the MMTV/v-Ha-ras transgene developed tumors earlier and at higher multiplicities in the absence than in the presence of p21Cip1. To further evaluate the combinatorial role of genetic alterations and loss of cell cycle inhibitors in tumorigenesis, we performed two companion studies. In the first study, wild type and p21Cip1-null mice were exposed to the chemical carcinogen, urethane. Similar to its effects in v-Ha-ras mice, loss of p21Cip1 accelerated tumor onset and increased tumor multiplicity in urethane-treated mice. Lung tumors were the predominant tumor type in urethane-treated mice regardless of p21Cip1 status. In the second study, tumor formation was monitored in v-Ha-ras mice expressing or lacking p27Kip1. Unlike p21Cip1, the absence of p27Kip1 had no effect on the timing or multiplicity of tumor formation, which was largely restricted to mammary and salivary glands. However, once tumors appeared, they grew faster in p27Kip1-null mice than in p27Kip1-wild type mice. Increases in growth rate were particularly striking for salivary tumors in ras/p27−/− mice. Loss of p21Cip1, on the other hand, had no effect on tumor growth rate in v-Ha-ras mice. Collectively, our data suggest that p21Cip1 suppresses tumor formation elicited by multiple agents and that p21Cip1 and p27Kip1 suppress tumor formation in different ways.

Identification and analysis of a collagenolytic activity in Streptococcus mutans.

Abstract.Streptococcus mutans is an important pathogen in coronal caries and is implicated in dental root decay by its ability to bind collagen from various sources. In the present study, electron microscopic analysis demonstrated the ability of S. mutans to bind and to disrupt collagen fibrils of the amniotic membrane. The synthetic peptide FALGPA, which is similar in structure to collagen, was degraded by S. mutans, with a lower level of FALGPA hydrolytic activity observed in sucrose-grown cells compared with cells grown in the absence of sucrose. Inhibition studies of FALGPA hydrolytic activity showed a pattern characteristic of collagenase activity, with inhibition by 1,10-phenanthroline and EDTA, but not by phenylmethylsulfonyl fluoride (PMSF). Additionally, immunological cross-reactivity was observed between proteins from disrupted cells of S. mutans and antiserum to collagenase from Clostridium histolyticum. Gelatinolytic activity was demonstrated by gelatin zymogram analysis. These findings suggest that collagenolytic activity by S. mutans may be an important virulence factor in dental root decay.

Src kinase induces tumor formation in the c‐SRC C57BL/6 mouse

Src kinase has been linked as a causative agent in the progression of a number of cancers including colon, breast, lung and melanoma. Src protein and activity levels are increased in colorectal cancer and liver metastases arising secondary to colon cancer. However, although Src protein is increased in colon cancer as early as the adenomatous polyp stage, a role for Src in carcinogenesis has not been established. We developed the c‐SRC transgenic mouse in the C57BL/6 strain to address the issue of carcinogenesis in cells with high levels of Src expression. The transgene was constructed with the human c‐SRC gene downstream of the mouse metallothionein promoter to create zinc inducible gene expression. In these C57BL/6 mice, Src protein was increased in a number of tissues both with and without zinc induction. No additional carcinogenic agent was administered. After 20 months, mice were assessed for tumor development in the liver and GI tract, as well as other organs. Of the mice with the transgene, 15% developed tumors in the liver while no tumors were detected in wild type C57BL/6 mice. A further study was conducted by crossing c‐SRC C57BL/6 mice with p21 nullizygous mice to determine the effect of oncogene expression combined with inactivation of the tumor suppressor gene, p21. Addition of the c‐SRC transgene to the p21−/− background increased tumor formation almost 3‐fold, while it increased metastasis 6‐fold. The data from our study show, for the first time, that Src kinase may play a role in carcinogenesis.

Purification and assay of bacterial collagenases

Many different types of bacteria, including pathogens, produce collagenolytic enzymes. In the past, it has been diffucult to determine the role of these enzymes in host-parasite relationships because few detailed purification procedures and collagenase-specific assays have been available. The development of procedures to purify bacterial collagenases to homogeneity and assays to specifically determine their collagenolytic activity have made it possible to further study and characterize these enzymes. Assays to measure collagenolytic activity include radioactive assays to measure of [14C]amino acids or peptides from radioactively labeled collagen, a viscometric assay to determine decreases in viscosity of collagen solutions, ninhydrin-based assays to detect amino acids and peptides liberated from collagen, synthetic peptide assays to measure hydrolysis of synthetic peptides similar in structure to collagen, and collagen film/gel assays to detect collagen hydrolysis. This paper reviews some of the techniques available to purify and assay bacterial collagenases.

Modified FALGPA assay for cell-associated collagenolytic activity

A continuous spectrophotometric assay monitoring the hydrolysis of the synthetic peptide 2-furanacryloyl-l-leucylglycyl-l-prolyl-l-alanine (FALGPA) is used to measure collagenase activity of both bacterial and vertebral collagenases. In the present study, a protocol was developed to adapt this assay to the measurement of cell-associated FALGPA hydrolytic activity in bacteria. The bacteria tested included Bacillus cereus, Streptococcus agalactiae, Streptococcus mutans, Enterococcus faecalis, and Escherichia coli, and various levels of activity were identified. The method presented here allows the detection of FALGPA hydrolysis using a small quantity of cells without the need for prior purification of the collagenolytic enzyme or collection and concentration of a large volume of culture supernatant fluid.

Degradation of amniotic collagen fibrils by group B streptococci

Amniotic membranes collected after both cesarean and vaginal deliveries were inoculated with group B streptococci (GBS) in this in vitro study. Transmission electron microscopic examination of segments of uninoculated control amniotic membranes revealed compact, wellordered, clearly defined layers of collagen fibrils. Examination by both scanning and transmission electron microscopy of amniotic membrane segments inoculated in vitro with group B streptococci revealed bacterial attachment to the membrane surface and migration through the membrane accompanied by disordered collagen fibril layers. Degradation of the collagen fibrils during bacterial invasion may cause weakening of the amniotic membranes and thus be a contributing factor in cases of premature rupture of membranes associated with group B streptococcal colonization of the mother.