Overview of matrix metalloproteinase expression in cultured human cells

Overview of Matrix Metattoproteinase Expression in Cuttured Human Ceils TROY A. GIAMBERNARDI*, GEORGEM. GRANT*, GAIL P. TAYLOR*, ROBERTJ. HAY'[, VERONICA M. MAHER~, J. JUSTIN MCCORMICK:~and ROBERTJ. KLEBE* * Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, Texas, t American Type Culture Collection, Rockville, Maryland and $ Carcinogenesis Laboratory, Departments of Microbiology and Biochemistry, The Cancer Center, Michigan State University, East Lansing, Michigan, USA

Abstract The matrix metalloproteinases (MMP) have been implicated in tumor invasion and metastasis both by immunohistochemical studies and from the observation that specific metalloproteinase inhibitors block tumor invasion and metastasis. Oligonucleotide primers for thirteen MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16) were optimized for use in RT-PCR. A semi-quantitative RT-PCR assay was used to determine the pattern of MMP mRNA expression in 84 normal and transformed or carcinogen transformed human cell lines and strains derived from different tissues. The results demonstrate one or more cell lines which express thirteen members of the MMP family. In addition, various oncogene transfected human fibroblast cell strains were analyzed for MMP expression. We confirm that over-expression of the H-ras oncoprotein correlates with up-regulation of MMP-9 and demonstrate that over-expression of v-sis also up-regulates MMP-9. A cell line immortalized following myc expression was found to up-regulate MMP-7, MMP-11 and MMP-13. Inappropriate expression of several MMP mRNAs was detected in breast, prostate, bone, colon and oral tumor derived cell lines. Identification of at least one cell line expressing each of thirteen MMPs and the observation of oncogene induced expression of several MMPs should facilitate analysis of the transcriptional mechanisms controlling each MMP. Key words: collagenases, gelatinases, matrix metalloproteinases, oncogenes, stromelysins.

Introduction The currently described matrix metalloproteinase (MMP) family consists of fifteen zinc containing proteases which cleave one or more components of the ex-

Abbreviations used: MMP, matrix metalloproteinase. Matrix Biology Vol. 16/1997/98, pp. 483-496 © 1998 by Gustav Fischer Verlag

tracellular matrix. Due to the overlapping specificity of the MMPs and the fact that MMPs are elaborated in a latent form, it is difficult to assay for individual MMPs by enzymatic methods. In this study, we describe RTPCR primer sets which can detect with high sensitivity mRNA for 13 of the 15 currently described members of the MMP family. Since many recently described members of the MMP family have not been identified in cul-

484

T.A. Giambernardi et al.

tured cells, we have used the primer sets described to detect permanent cell lines which express each of the MMPs. This study corroborates many prior protein level studies of M M P expression in cell lines. The primer sets described and identification of cell lines expressing most members of the M M P family should expedite future analysis of the regulation of M M P gene expression. The results presented indicate the ubiquitous expression of m R N A for two of the membrane-type MMPs (MMP-14 and MMP-15). Most fibroblastic cell lines expressed high levels of the m R N A s for M M P - 1 , -2, -3, -7, -10, -14, -15 and -16; hence, examination of tissues would be problematic. Since coordinate expression of any pair of M M P s was not observed, it is likely that each member of the M M P family is regulated by independent control mechanisms. Analysis of the promoters of several of the M M P s indicates that some of the M M P s are regulated by o n t o proteins (Schonthal et al., 1988; Hagmeyer et al., 1993). Thus, there is accumulating evidence that the expression of M M P s changes during the progression of cells toward malignancy (Ray and Stetler-Stevenson, 1994; Hopkin, 1996). This study provides additional evidence that changes in the regulation of M M P genes occurs during the evolution of tumor cells toward a more malignant phenotype. For example, it is demonstrated that more invasive m a m m a r y carcinomas and prostate tumors express more members of the M M P family than do more benign tumors. Secondl), it is shown that over-expression of oncogenes can up-regulate expression of several MMPs. One of the difficulties in analyzing the carcinogenic process is that although there are m a n y malignant cell lines available that were derived from h u m a n or animal tumors, the normal cell that ultimately gave rise to these tumors is generally unavailable. For this reason, we have included in the present study a h u m a n fibroblast cell lineage in which both the normal skin fibroblast progenitor and oncogene transformed derivatives are included. By sequential clonal selection, McCormick and his colleagues (Morgan et al., 1991; McCormick and Maher, 1996) have derived a series of non-tumorigenic cell strains that have acquired, in a stepwise fashion, various characteristics exhibited by h u m a n fibroblastic t u m o r cells. The ultimate cell strains in this lineage form highly malignant tumors in athymic mice. In this system, we demonstrate that expression of specific M M P s is altered during conversion of normal fibroblasts into cells capable of forming malignant tumors.

Materials and Methods Cell culture

Before total R N A extraction, cultures were grown to 80% confluence in 50% Dulbecco's M E M / 5 0 % H a m ' s F-12 containing 10% newborn calf serum plus 100 units/ml penicillin, 100 mg/ml streptomycin and 50 pg/ml gentamicin. With the exceptions noted below, all cell lines were obtained from the American Type Culture Collection, Rockville, MD. Cell lines T H P - I and HL-60 were a gift from Dr. A n t h o n y Valente of the Uni-

~--

H-ras ~

L98--v-fe8

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---H-ras

~: PH.aM CL141.JM13

Oncogene

--N-ras

....

. . DW.3P

Transformants

DW.5P

--v-K-ras . . . . . .

DFL95.4B

--v-sis

LG1

v.myc

*DY620.10C3

*r~T,

MSU-1.0~MSU-I.1

e

, CCL32.3A

~vlmu-1.2 ~ v - m s < : . CCL53.26B DY616.3CI BPDE .

.

.

. DY616.4C5

Carcinogen Transformants

--g-radiation .

', ~VV-radiation

.

.

.

MW7.3A2

. SWKI21.B5 ~- + SWKI21.CI6 SVgKI22.BI1

Figure l. This figure describes schematically the origin of tile MSU1 family of cells. The backbone of the lineage are the MSU-1.0, MSU-I.I, and MSU-1.2 cells stains which are nontumorigenic. Each cell strain in the backbone is more transformed than the cells from which it is derived. All of the cell strains derived from MSU-I.I and MSU-1.2 are tumorigenic, with the exception of L98. When a "T" is appended to the name, it indicates that the cells were derived from a tumor which formed in an athymic mouse by injection of the cells. The following, well characterized cell lines and strains were employed: LGI (Morgan et al., 1991), MSU-I.0 (Morgan et al., 1991), MSU-I.1 (Morgan et al., 1991), MSU-1.2 (I,in et al., 1995), CCI,83.1 (Lin et al., 1995), CCL140.14A (Linet al., 1994), PH.2M (Hurlin et aI., 1989), PH.3M (Hurlin et al., 1989), DW.3P (Wilson et al., 1990a), DW.5P (Wilson et al, 1990a), DFI,95.4B (Fry et al., 1990), DY620.10C3 (Yang et al., 1992), 1,98 (Lin et al., 1994),CCL32.3A (Senoir et al., 1996) and CCI,53.26B (Senoir et al., 1996). The I)Y616.3CI and DY616.3C1 strains were obtained by treannent of the MSU-I.I cells with an activated form of benzola]pyrene (BPDE), selection for foci, and isolation of clonal populations (Yang et al., 1992). The CLI41.JMI3 cell line was obtained by transfection of the H-ras (T24) oncogene with an MT prorooter into MSU-I.1 cells. The MWT.3A2 cell strain was obtained by g-irradiation of MSU-1.1 cells, selection for loci, and isolation of a clonal population. The SWKI21.BS, SWK121.C16 and SWK122.BI 1 cell strains were obtained in a similar fashion by UV-irradiation of MSU- I. 1 cells.

MMP Overview versity of Texas Health Science Center, San Antonio, TX. LG1 and its derivatives (Table IV) were established by Dr. J.J. McCormick and co-workers, Michigan State University, E. Lansing, MI (see Fig. 1 for details).

RT-PCR Total RNA was isolated from cultured cells by adding a guanidine thiocyanate solution (solution D) directly into the culture vessel (Chomczynski and Sacchi, 1987; (;rant et al., 1996). M M P and aldolase m R N A levels were determined by RT-PCR as previously described (Milam et al., 1991; Chen et al., 1992; Steffensen et al., 1992). In this report, we present a series of validated PCR primer sets that can be used in analyzing 13 members of the M M P family. The sequences of the PCR primers introduced in this study are given in Table I. The primer sets were validated by cycle sequencing with a d s DNA Cycle Sequencing System (BRL Life Technology, Gaithersburg, MD). The primer sets described for the MMPs yield one band of the predicted size for each member of the M M P family. PCR primer sets for aldolase (Chen and Klebe, 1993) and the other MMPs have been reported previously (Grant et al., 1996). As expected, false priming can generate spurious PCR products of many sizes following an excessive number of PCR cycles. This study provides positive control cell lines which make each of the MMPs (Tables II and III). We suggest that a known positive and negative cell line be used as a control in studies with new cells or tissues to ensure that the PCR conditions employed are correct. All of the studies described here were carried out with cell lines grown to semi-confluence in 10% newborn calf serum. If serum-free medium or medium containing fetal calf serum were employed, we expect that the expression of MMPs might change due to the known influence of peptide growth factors on M M P expression (BirkedalHansen, 1995). The RT-PCR assay was performed in 0.2 ml tubes under optimized conditions that employ standardized, unit-of-use stock solutions as previously described (Chen et al., 1993). Briefly, a 10 tll reverse transcription reaction was carried out at 42 °C for 60 min using 0.5 lag total RNA, 5.3 lal DEPC-treated H20, 0.5 I~1 200 ng/~l antisense primer, and 3.2 I~1 AMV mix (0.5 lal of 1 M Tris, pH 8.3; 0.4 lal of 1 M KCL; 0.8 lal of 0.1 M MgC12; 0.5 pl of 5 mg/ml BSA; 0.25 lal of 0.5 M DTT; 0.25 lal of 40 U/1 RNasin; 0.2 t~1 of 25 m M dNTP; 0.025 ml of 20 U/ml AMV reverse transcriptase; and 0.275 lal of DEPC-treated H20). The PCR step was performed in a final volume of 50 lal which consisted of

485

Table I. Primer sets for MMP analysis. Based on previously reported mRNA sequences in the GenBank repository, sense and antisense oligonucleotide primers ranging from 21 to 30 nt were designed with the Oligo primer program (Version 4.06). The primer sets were synthesized by Genosys Biotechnologies, Inc. (Woodlands, TX). Only those primer sets which produced a single PCR product of the predicted size were used. The product size and optimal re-annealing temperature are presented for each primer set. Primer sequences for MMP-1, MMP-2, MMP-7, MMP-8, MMP-9 and MMP-10 were previously described (Grant et al., 1996). MMP-1 (Fibroblast collagenase) (Product size = 786 bp, 58 °C) 5"-CGACTCTAGAAACACAAGAGCAAGA-3" (sense) 5"-AAGGTTAGCTTACTGTCACACGCTT-3" (antisense) MMP-2 (72 kDa Gelatinase) (Product size = 605 bp, 58 °C) 5"-GTGCTGAAGGACACACTAAAGAAGA-3" (sense) 5"-TTGCCATCCTTCTCAAAGTTGTAGG-3" (antisense) MMP-3 (Stromelysin-1) (Product size = 729 bp, 55 °C) 5"-GAACAATGGACAAAGGATACAACA-3" (sense) 5"-TTCTTCAAAAACAGCATCAATCTT-3" (antisense) MMP-7 (Matrilysin) (Product size = 373 bp, 58 °C) 5"-GGTCACCTACAGGATCGTATCATAT-3" (sense) 5"-CATCACTGCATTAGGATCAGAGGAA-3" (antisense) MMP-8 (Neutrophil collagenase) (Product size = 435 bp, 51 °C) 5"-GCTGCTTATGAAGATTTTGACAGAG-3" (sense) 5"-ACAGCCACATTTGATTTTGCTTCAG-3" (antisense) MMP-9 (92 kDa Gelatinase) (Product size = 243 bp, 58 °C) 5"-CACTGTCCACCCCTCAGAGC-3" (sense) 5"-GCCACTTGTCGGCGATAAGG-3" (antisense) MMP-10 (Stromelysin-2) (Product size = 408 bp, 58 °C) 5"-CACTCTACAACTCATTCACAGAGCT-3" (sense) 5"-CTTGGATAACCTGCTTGTACCTCAT-3" (antisense) MMP-11 (Stromelysin-3) (Product size -- 326 bp, 58 °C) 5"-TAAAGGTATGGAGCGATGTGAC-3" (sense) 5"-TGGGTAGCGAAAGGTGTAGAAG-3" (antisense) MMP-12 (Metalloelastase) (Product size = 517 bp, 58 °C) 5"-TTCCCCTGAACAGCTCTACAAGCCTGGAAA-3" (sense) 5"-GATCCAGGTCCAAAAGCATGGGCTAGGATT-3" (antisense) MMP-13 (Collagenase-3) (Product size -- 330 bp, 51 °C) 5"-GTGGTGTGGGAAGTATCATCA-3" (sense) 5"-GCATCTGGAGTAACCGTATTG-3" (antisense) MMP-14 (Membrane-type-1 MMP) (Product size = 497 bp, 62 °C) 5"-CGCTACGCCATCCAGGGTCTCAAA-3" (sense) 5"-CGGTCATCATCGGGCAGCACAAAA-3" (antisense) MMP-15 (Membrane-type-2 MMP) (Product size = 454 bp, 62 °C) 5"-ACAACCACCATCTGACCTTTAGCA-3" (sense) 5"-AGCTTGAAGTTGTCAACGTCCTTC-3"(antisense) MMP-16 (Membrane-type-3 MMP) (Product size = 652 bp, 58 °C) 5"-TTACTTCTGGCGGGGCTTGCCTCCTAGTAT-3" (sense) 5"-ACAGTACAGTATGTGGCGGGGTGTTCCTTT-3" (antisense)

Description

Breast, human Breast, adenocarcinoma Breast, ductal carcinoma Mammary epithelial Breast, ductal carcinoma Breast, adenocarcinoma MCF-7 invasive variant Breast, adenocarcinoma Breast, ductal carcinoma Breast, adenocarcinoma Breast, adenocarcinoma Breast, adenocarcinoma Breast, ductal carcinoma Breast carcinoma

Prostate carcinoma Prostate carcinoma Prostate carcinoma

Osteosarcoma Osteosarcoma Osteosarcoma, primary

Duodenum, adenocarciuoma Colon adenocarcinoma Colon adenocarcinoma

Oral, carcinoma Oral, carcinoma

Embryonal carcinoma Embrvonal carcinoma

Cell Line

Mammary H B L - 100 BT-20 BT-549 MCF-10F Hs 578T MCF-7 MCF-7M/Adr MDA-MB-231 MDA-MB-435S MDA-MB-436 MDA-MB-468 SK-BR-3 T-47D ZR-75-1

Prostate LNCap DU 145 PC-3

Bone HOS MG63 Saos-2

Gastrointestinal HuTu 80 SW480 SW620

Oral A253 KB

Embryonal Tera- l Tera-2

1

MMP

I+

0

0 0

1+ 5+"'

0 0

0

4+ 01

0

1+

1+' 5+' 5+ k

0 0 l+h

0

4+ I 5+~ 0+ k

0 0 5+

4+

5+

5+ '~ Oc 2+ t' I+ ~' 0a 5+ ~ 0b 0 Ob 0b

1+

0 0 5+ 5+ 5+ 5+ 0 0 0 0

0 0

0 0

0

0

0

I+ 5+ 1+

0 0 I+

0 0 0 0 5+ 4+ 0 0 0 0

0 0 1+

3

0 0 5+ '~

0 0 5+

MMP 2

MMP

4+ 5+

4+ 5+

4+

4+

I+

0 0 4+

5+~ 1+ 4+

1+ 0 0 1+ 1+ 4+ 5+ 4+ 1+ 3+

5+

2+ 0 0

7

MMP

0 0

0 0

0

0

0

0 0 0

0 0 0

0 0 0 0 0 0 0 0 0 0

0

0 0 0

8

MMP

0 ()"

0 01

1+

5+

0

1+ ~ 0 0

0 0 1+

() 0 1+~' t+ '1 I+' I+ 1+ 1+ 0 0

0

0 0 0

9

MMP

0 5+

3+ 0

2+

0

5+

0 4+ 0

0 0 3+

0 0 1+ 1+ 1+ 3+ 1+ I+ 3+ 1+

5+

0 0 0

10

MMP

0 1+

0 0

1+

0

0

0 0 0

0 0 0

0 0 0 0 0 0 0 0 0 0

0

0 0 0

11

MMP

0 0

0 0

0

0

0

0 0 0

0 0 0

0 0 1+ 0 1+ 0 0 0 0 0

5+

0 0 1)

MMP 12

4+ 4+

I+ 0

,5+

2+

I+

0 0 3+

0 0 1+

0 0 0 0 0 0 0 0 0 0

0

0 0 0

MMP 13

5+ 5+

5+ 0

4+

5+

5+

5+ 5+ 5+

0 .5+ 5+

5+ I' 0 t' 5+ 5+ ~' 5+ t' 5+ 5+ 0 2+ 1+

5+

5+ 1+ 5+ >

MMP 14

5+ 5+

4+ 5+

.5+

4+

5+

4+ 5+ 3+

.5+ .5+ 5+

3+ 5+ 5+ 4+ 5+ 5+ 5+ 5+ 5+ 5+

4+

4+ 5+ 5+

MMP 15

3+ 5+

0 0

0

0

0

3+ 0 4+

[+ 0 5+

5+ 4+ 4+ 0 5+ 5+ 0 0 i+ 1+

5+

5+ 0 5+

MMP 16

Table II. Comparison of M M P expression in tumors derived from the same or similar cell type. Cell lines derived from human tumors are grouped together according to their tissue of origin and tumor type. As indicated in the text, 5+ indicates that strong expression was observed (PCR product obtained between PCR cycles 20-25), and 1+ indicates the lowest level of expression. If no expression was observed by PCR cycle 45, a "0" was entered. The results indicate that tumors derived from the same cell type can express different repertoires of M M P mRNAs. The references, given as superscript letters, provide corroboration at the protein and/or RNA level for the RT-PCR results presented. The references presented in this table are as follows: a = Azzam et al., 1993, b = Pulyaeva et al., 1997; c = Frisch and Morisaki, 1990; d = Liu and Rose, 1995; e = Haupt et al., 1996; f = Rose et al., 1994; g = Sundareshan et al., 1997; h = Stearns and Wang, 1994; i = Panagakos and Kumar, 1994; j = Masure et al., 1990; k = Tsuchiya et al., 1994; 1 = Shmdoh et al., 1996; m = Tienari et al., 1994; n = Mackay et al., 1992.

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E.

C~

>

o~

Description

Fibrosarcoma Muscle, rhabdomyosarcoma

Promyelocytic leukemia Monocytic leukemia Histocytic lymphoma

Lung, carcinoma Lung, carcinoma, bronchogenic Brain, astrocytoma Brain, glioblastoma Placenta, choriocarcinoma Cervical carcinoma Hepatocellular carcinoma Melanoma Melanoma Kidney, leiomyoblastoma Adrenal cortex, adenocarcinoma Thyroid carcinoma

Cell Lines

Sarcomas HT-1080 RD

Leukemias HL-60 THP-1 U937

Carcinomas A549 ChaGo K-1 CCF-STTG 1 T98G JEG-3 HeLa Hep G2 A375 G-361 G-402 SW-13 SW-579 I+ 0 5+ 2+ 0 0i 0 3+ 0 1+ 4+ 5+

0 1+¢ 0~

5+" 0

MMP 1

1+I' 0 5+ I 5+ I l+ Ok 0 5+ m 5+ 0 0 5+

1+ 4+ a 0

5+" 0

MMP 2

1+ 0 5+ 1+ 0 0 1+ 2+ 0 4+ 0 4+

0 0 0

1+~ 0

MMP 3

5+ 0 5+ 4+ 0 4+ 5+ 1+ 0 0 5+ 4+

5+ 3+ 0

5+ 0

MMP 7

0 0 0 0 0 0 0 5+ 4+ 0 0 0

0 0 0

0 0

MMP 8

0" 0 0 0 01 0 0 0 0 0 0 0

5+ b 5+ " 1 +,~

1+ ~ 0

MMP 9

1+ 0 0 0 0 0 0 4+ 1+ 1+ 5+ 0

0 I+ 1+

0 0

MMP 10

0 0 5+ 0 0 0 5+ 0 0 0 0 1+

0 0 0

0 0

MMP 11

0 0 1+ 0 I+ 0 0 0 1+ 0 0 0

0 0 0

0 0

MMP 12

0 0 0 1+ 0 0 1+ 0 0 0 4+ 0

0 0 0

0 0

MMP 13

1+ 4+ 5+ 5+ 5+ 4+ 4+ 5+ 5+ 4+ 3+ 5+

4+ 5+ 4+

5+' 5+

MMP 14

4+ 5+ 5+ 1+ 5+ 5+ 5+ 5+ 4+ 4+ 5+ 4+

0 5+ 5+

4+ 3+

MMP 15

0 0 5+ 3+ 0 0 0 5+ 1+ 1+ 1+ 5+

0 0 0

5+ 1+

MMP 16

Table III. MMP expression in cells of different tumor type. Cell lines derived from human tumors are grouped together according to their tumor type. Expression levels of MMP mRNAs are presented as described in the text and Table II. The references provided are to studies conducted at the protein and/or RNA levels. The references, given as superscript letters, provide corroboration at the protein and/or RNA level for the RT-PCR results presented and are as follows: a = Lohi et al., 1996, b = Ries et al., 1996; c = Lacraz et al., 1994; d = Chang et al., 1996; e = Houde et al., 1996; f = Callaghan et al., 1996; g = Weston and Weeks, 1996; h = Zucker et al., 1992; i = Abe et al., 1994; J = Lambert et al., 1993; k = Frisch and Morisaki, 1990; I = Graham et al., 1994; m = Monsky et al., 1994; n = Mackay et al., 1992.

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T.A. Giambernardi et al.

10 lal of the above reverse transcription reaction + 1 lal 100 ng/lal sense primer + 39 pl of Taq mix (3.2 !ul of 500 mM KCL; 0.8 tJ1 of 100 mM Tris, pH9; 2.4 lal of 25 mM MgCI2; 0.4 1~1 of 25 mM dNTP; 0.25 !ul of 5 U/lal Taq polymerase; and 31.95 pl of DNase-free H20). Unless stated otherwise, the standard cycling conditions used were as follows: initial denaturation at 94 °C for 30 s, annealing at optimal Tm (see Table I) for 1 rain, elongation at 72 °C for 1 rain 50 s, and 94 °C for 20s.

Comparison of RNA levels To compare MMP mRNA levels in different cell lines, results were standardized with respect to the constitutive enzyme, aldolase, as previously described (Chen and Klebe, 1993). Briefly, the amount of total RNA used in each assay was adjusted such that each RNA sample had an identical amount of aldolase mRNA. Standardization was carried out by performing RT-PCR with ~2P-endlabeled aldolase primers. All samples were amplified to PCR cycle 16, which is in the exponential range of amplification for aldolase. Following separation on 5% polyacrylamide gels (PAGE), PCR product bands were visualized by ethidium bromide staining, and the radioactivity in each band was determined by scintillation counting. If the amount of radioactivity in PCR products derived from a given cell line was not comparable to other cell lines, the amount of total RNA employed was increased (or decreased) such that equal amounts of radioactivity in aldolase PCR products was obtained. Hence, in analysis of MMP gene expression, amounts of total RNA which yielded comparable levels of aldolase mRNA were employed.

Semi-quantitative analysis of MMP expression Initially, total RNAs from all cell lines were amplified for 45 PCR cycles with primer sets specific for given members of the MMP family. If no PCR product was observed at PCR cycle 45 after two RT-PCR repetitions, the cell line was considered to be negative for the MMP in question. Those cell lines positive for a given MMP at cycle 45 were amplified again, and 15 lal aliquots were withdrawn at PCR cycles 25, 30, 35 and 40. The cycle at which a PCR product could first be detected was determined. The results of this analysis are presented in Tables II-IV as follows: cycle 20-25 = 5+ (highest level of expression); cycle 26-30 = 4+; cycle 31-35 = 3+; cycle 36-40 = 2+; cycle 41-45 = 1+ (lowest level of expression); 0 = no expression noted after 45 cycles.

Resutts and Discussion PCR primer sets for the MMP family We have developed PCR primer sets which yield a single band for given members of the MMP family (Table I). Each primer set has been characterized (a) by demonstration that it generates a PCR product of the predicted size and (b) by sequence analysis of the PCR product (data not shown). These primer sets should be of considerable value in future analysis of MMP gene expression. Analysis of MMPs at the protein level is quite difficult, because the overlapping substrate specificity of MMPs (Birkedal-Hansen, 1995) makes it difficult to distinguish between different MMPs solely by enzymatic means. In addition, MMPs are often elaborated by cells in latent forms which require special treatments to yield an active enzyme (VanWart and BirkedalHansen, 1990). By an extensive literature survey, it can be shown that the RT-PCR results presented here corroborate prior studies of MMP expression that were carried out using various techniques at the protein and/or RNA levels (see references in parentheses in Tables II-IV). In addition to corroborating reports of expression of given MMPs in certain cell lines, it is noteworthy that earlier reports of the lack of expression of MMPs could also be verified in this report (Tables lI-III). The simplicity and sensitivity of the RT-PCR assays presented should expedite future analysis of the expression of MMPs.

Semi-quantitative comparison of gene expression The RT-PCR assays developed here were used to analyze the expression of MMPs in 84 cell lines or strains. A semi-quantitative method was employed to compare expression of MMPs in different cell lines. First, results were standardized by adjusting the amount of total RNA used per assay such that the RNA sample for each cell line yielded comparable levels of aldolase RT-PCR product at cycle 16. Second, as described in detail in the methods section, mRNAs were assayed over a range of 25 PCR cycles in order to compare MMP expression in different cell lines. We have found that expression of MMP mRNA in different cell lines can vary by as much as 25 PCR cycles under standardized conditions. While the methods employed here cannot detect small differences in gene expression, no difference is discussed which resulted from less than a ten PCR cycle difference between two cell lines.

5+ 5+ ~ 4+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+

SV-40 Transformed Lung Fibroblast Sv-IMR-90 SV-40 transformed SV-MRC-5 SV-40 transformed SV-WI38 SV-40 transformed

Normal Dermal Fibroblast LG1 Normal, skin

Transformed LG1 Dermal Fibroblast MSU-1.0 v-myc MSU-I.1 v-myc, 45 chromosomes MSU-1.2 v-myc, PDGF-b expression CL141.JM13/T v-myc + H-ras, tumor derived PH.2M v-myc + H-ras PH.2MT v-myc + H-ras, tumor derived PH.3M v-myc + H-ras PH.3MT v-myc + H-ras, tumor derived L98 v-myc + low expression H-ras CCL83.1 v-myc + H-ras + v-fes CCL83.1/T v-myc+ H-ras + v-fes, tumor derived CCL140.14A v-myc + H-ras + v-fes CCL140.14A/T v-myc + H-ras + v-fes, tumor derived DW.3P v-myc + N-ras DW.3P/T v-myc + N-ras DW.SP v-myc + N-ras DW.5P/T v-myc + N-ras DFL95.4B v-myc + v-K-ras DFL95.4B/T1 v-myc + v-K-ras, tumor derived DFL95.4B/T2 v-myc + v-K-ras, tumor derived DY620.10C3/T v-myc + v-sis, tumor derived CCL32.3A v-myc + v-fes CCL32.3AFF v-myc + v-fes, tumor derived CCL53.26B v-myc + v4es DY616.3C1/T v-myc + Benzo, tumor derived DY616.4C5/T v-myc + Benzo, tumor derived MW7.3A2/T v-myc + g-irrad, tumor derived SWK121.B5 v-myc + UV SWK121.B5/T v-myc + UV, tumor derived SWK122B.11 v-myc + UV SWK122B.11/T v-myc + UV, tumor derived SWK121.C16 v-myctUV SWK 121.C16/T v-myc + UV, tumor derived

MMP 1

5+ 5+ 5+

Description

Normal Lung Fibroblast IMR-90 Normal MRC-5 Normal WI38 Normal

Cell Lines

5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5÷ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+

5+

4+ 5+ 4+

5+ ~' 5+ ~ 5+

MMP 2

5+ 5+ 5+ 5+ 5+ 4+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 4+ 4+ 4+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 1+ 1+

5+

0 5+ 4+

5+ 5+ 4+

MMP 3

5+ 5+ 5+ 4+ 4+ 5+ 5+ 4+ 4+ 3+ 4+ 3+ 2+ 5+ 5+ 4+ 5+ 3+ 5+ 3+ 4+ 5+ 3+ 3+ 4+ 4+ 3+ 5+ 5+ 5+ 5+ 4+ 4+

1+

5+ 3+ 3+

l+ 4+ 1+

MMP 7

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0

0 0 0

0 0 0

MMP 8

0 1+ 1+ 5+ 5+ 5+ 4+ 5+ 0 0 0 0 0 1+ 0 1+ 0 2+ 1+ 1+ 5+ 0 0 0 1+ 0 0 0 0 0 0 0 0

0

4+ b 0 0

0" 0a 0

MMP 9

4+ 5+ 0 4+ 5+ 5+ 5+ 3+ 4+ 4+ 4+ 5+ 4+ 5+ 4+ 5+ 1+ 5+ 4+ 5+ 5+ 5+ 4+ 3+ 3+ 3+ 0 5+ 0 3+ 4+ 0 0

5+

4+ 3+ 4+

4+ 3+ 4+

MMP 10

5+ 4+ 4+ 4+ 4+ 5+ 2+ 4+ 5+ 2+ 3+ 4+ 5+ 4+ 1+ 5+ 4+ 5+ 1+ 5+ 2+ 4+ 4+ 4+ 4+ 4+ 2+ 5+ 4+ 3+ 5+ 5+ 4+

2+

0 0 1+

0 0 0

MMP 11

0 0 0 0 0 0 4+ 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0

0 0 0

0 0 0

MMP 12

5+ 5+ 1+ 3+ 4+ 5+ 4+ 3+ 4÷ 1+ 1+ 1+ It 4+ 4+ 1+ 5+ 5+ 4+ 5+ 3+ l+ 1+ l+ 3+ 4+ 3+ 4+ 4+ 4+ 4+ 5+ 5+

1+

4+ 1+ 4+

2+ 1+ 0

MMP 13

5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+ 5+

5+

5+ 5+ 5+

5+ 5+ 5+

MMP 14

5+ 5+ 3+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 4÷ 3+ 3+ 5+ 4+ 4+ 5+ 3+ 4+ 4+ 3+ 4+ 4+ 4+ 4+ 4+ 4+ 4+ 3+ 5+ 5+

3+

2+ 4+ 2+

2+ 2+ 5+

MMP 15

3+ 5+ 3+ 3+ 3+ 4+ 3+ 4+ 5+ 5+ 4+ 5+ 5+ 4+ 5+ 3+ 5+ 3+ 5+ 5+ 4+ 5+ 5+ 5+ 4+ 4+ 4+ 4+ 5+ 4+ 3+ 4+ 3+

4+

1+ 5+ 1+

5+ 5+ 4+

MMP 16

Tablc IX,'. MMP expression in t r a n s [ o r m d tibroblasts. The bV40 transformed derivatives of the normal human fibroblast cell lines (WIng, IMR-90 and MRC-5) were compared to their normal counterparts. All of the transformed dermal fibroblast cell lines are derivatives of the karyotypically normal human dermal fibroblast line, LGI, and their derivation is described in Figure 1. Scoring of M M P expression is as presented in Table II. The transforming agent used to generate each cell strain is indicated. The references, given as superscript letters, provide corroboration at the protein and/or RNA level for the RT-PCR results presented and are as follows: a = Mackay et al., 1992; b = Wilhelm et al., 1989; c = Imai and Takano, 1992.

oo ~o

©

490

T.A. Giambernardi et al.

Comparison of expression of MMPs in permanent cell lines and cell strains Since the presence of fibroblasts, macrophages and other types of cells in virtually all tissues would result in the detection of many MMP mRNAs by RT-PCR, we chose not to examine tissues. For example, karyotypically normal fibroblasts (see WI-38, IMR-90, MRC-5 and LG1 in Table IV) all express the mRNAs for MMP1, MMP-2, MMP-3, MMP-7, MMP-10, MMP-14, MMP-15 and MMP-16; hence, RT-PCR analysis of most tissues would result in the observation of all of the above MMPs. Thus, if MMP expression in tissues is to be studied, immunohistochemical or in situ hybridization would need to be used to detect MMPs at the single cell level. Using the above RT-PCR assay, the pattern of expression of MMP mRNAs in 51 cell lines and 33 cell strains was assessed (Table If-IV). Many of the cells used in the survey are used in the National Cancer Institute Tumor Screen (Boyd and Paull, 1995) and are also readily available from the American Type Culture Collection.

Similarities in expression of MMPs in selected cell types Certain generalizations can be drawn from the study. First, all but three of the 84 cell lines examined expressed the mRNAs for two of the membrane-type MMPs (MMP-14 and MMP-15) (Tables II-IV). Second, the majority of the fibroblastic cell lines examined expressed the mRNAs for MMP-I, -2, -3~ -7, -10, -14, -15 and -16. The notable exceptions to these observations were the lack of MMP-3 mRNA expression in the SV40transformed line, SV-IMR-90, and the lack of MMP-10 mRNA expression in the fibrosarcoma line, HT-1080. Third, the osteosarcoma cells examined rivaled fibroblasts in the number of MMPs expressed (Table II). Fourth, while fibroblasts and osteosarcomas produce a large number of MMPs, non-mesenchymal cell lines generally produce few MMPs. For example, the relatively non-invasive mammary carcinoma MCF-7 (Thompson et al., 1992; Wang et al., 1996) and the non-invasive prostate carcinoma LNCaP (Hoosein et al., 1996; Leyton et al., 1996) produce only two or three MMPs, respectively (Table II). Fifth, we did not note the coordinate expression of any pair of MMPs. Except for the nearly ubiquitous expression of MMP14 and MMP-15, expression of a given MMP did not necessitate the expression of another. This observation would suggest that expression of each MMP is under independent control.

In the remainder of this section, we will discuss our findings for each major class of MMP, i.e., the collagenases, stromelysins, gelatinases and membrane-type MMPs.

Collagenases (MMP- 1, MMP-8, MMP- 13) Interstitial collagenases are the only members of the MMP family capable of cleaving fibrillar collagens. These collagenases cleave the native helix of type I, II and III fibrillar collagens at a single peptide bond, generating fragments approximately one quarter and three quarters the size of the original molecule (Hasty et al., 1996; Welgus et al., 1996). MMP-8 (neutrophil collagenase) mRNA expression was detected in both (;-361 and A375 melanoma cell lines studied. To our knowledge, this is the first report of any cell line expressing neutrophil collagenase other than primary neutrophils. The G-361 and A375 cell lines are good immortalized cell lines for analysis of MMP-8 gene regulation. It is possible that MMP-8 expression is a characteristic of melanoma cells. High level MMP-13 (collagenase-3) mRNA expression was found in 13 cell lines of different origins. A previous report (Freije et al., 1996) indicated that MMP-13 mRNA expression occurred only in mammary tumors. The results reported here suggest that further investigation of MMP-13 expression in other tumor types is warranted.

Stromelysins (MMP-3, MMP-7, MMP-10, MMP-11, MMP-12) Stromelysins have a broad substrate specificity and are able to degrade many extracellular proteins, including proteoglycans, laminin, elastin and fibronectin (Murphy et al., 1996; Quantin et al., 1996). MMP-7, which is found in most exocrine glands (Saarialho-Kere et al., 1995), was expressed by 32 of the 43 non-fibroblastic cell lines examined (Tables II-llI) as well as all of the fibroblastic cell lines and strains (Table IV). MMP-11 (stromelysin-3) expression has previously been reported in human breast tumors in the stromal cells immediately surrounding cancer cells but not in the cancer cells themselves (Basset et al., 1994). We confirm the finding of M M P - l l mRNA expression in human dermal fibroblasts (Table IV); however, we also report that high levels of MMP-11 mRNA are detected in certain non-fibroblastic cell lines, namely, CCF-STTG1 (astrocytoma) and HepG2 (hepatoma) and, at low levels, in MDA-MB-231 (breast adenocarcinoma), SW620 (colon

MMP Overview adenocarcinoma), SW-579 (thyroid carcinoma) and Tera-2 (teratoma) (Tables II-III). MMP-12 expression was previously noted in human placenta tissue (Belaaouaj et al., 1995) and activated macrophages (Shapiro et al., 1993). MMP-12 was expressed at a high level in MCF-10F (Table II). We detected low levels of MMP-12 (metalloelastase) expression in CCF-STTG1 (astrocytoma), G-361 (melanoma) and the JEG-3 (choriocarcinoma) cell lines (Table IIl). The JEG-3 cell line, which is of placental origin, did express MMP-12; however, MMP-12 was not observed in several lymphocytic cell lines (HL-60, THP-1 or U937).

Gelatinases (MMP-2, MMP-9) Gelatinases degrade types IV, V, VII and X collagens, elastin and fibronectin, and they may act synergistically with interstitial collagenases in the degradation of fibrillar collagens (Fessler et al., 1984; Senoir et al., 1996; Wilhelm et al., 1996). The gelatinases differ structurally from the other MMPs by having a fibronectin type II insert in the catalytic domain. MMP-2 was expressed by 22 of the 43 non-fibroblastic cell lines examined (Tables II-III) and all of the fibroblast cell lines and strains studied (Table IV). MMP-9, which is expressed by only 13 of the 43 nonfibroblastic cell lines studied, is discussed below in regard to its control by oncoproteins.

Membrane-Type MMPs (MMP-14, MMP- 15, MMP- l 6) Membrane-type MMPs (MT-MMPs) are unique in that they possess a membrane-spanning sequence in the fourth pexin-like repeat of the C-terminal domain. In contrast to other MMPs, the MT-MMPs are not secreted. MMP-14 has been reported to activate MMP-2 (gelatinase A) (Sato et al., 1994; Cao et al., 1996). While Okada et. al. (1995), using Northern analysis, reported that MMP-14 mRNA expression was found mainly in fibroblastic cell lines, we found that MMP-14 as well as MMP-15 mRNA expression occurred in the vast majority of cell lines examined. We confirm their report that MMP-14 mRNA expression occurs in the MDA-MB-231 and HBL-100 cell lines; however, contrary to this report, we find expression of MMP-14 mRNA in both HL-60 and HeLa cells. The differences between our findings and those of Okada et al. may be due to the fact that RT-PCR is a more sensitive assay than Northern blotting.

491

Oncogenes control expression of MMPs Recent progress in the analysis of MMP promoter regions has revealed that several proto-oncogenes control MMP gene expression. Transactivation of the MMP-3 gene has been shown to involve members of the following proto-oncogene families: jun, fos, ets, myb and p53 (Buttice and Kurkinen, 1993; Buttice and Kurkinen, 1994). The MMP-1, MMP-7 and MMP-9 genes also possess AP-1 and PEA-3 sites for jun, fos and ets (Gutman and Wasylyk, 1990; Gaire et al., 1994; Higashino et al., 1995). In addition, MMP-9 also has an RB GT-box as well as SP-1 and NF-~cB sites (Sato et al., 1993; Sato and Seiki, 1993). Thus, it is not surprising that the expression of MMPs is altered following changes in oncogene expression. In the following section, we discuss effects of oncogenes on MMP expression noted in this study.

Possible effect of myc on MMP expression The normal and transformed dermal fibroblast cell lines and strains examined in Table IV were previously described and characterized in regard to their transformation and tumorigenicity properties (Wilson et al., 1990; Morgan et al., 1991; Yang et al., 1992; Lin et al., 1994; Yang et al., 1994; Lin et al., 1995; McCormick et al., 1995; McCormick and Maher, 1996). As shown in Figure 1, the backbone of the MSU1 lineage consists of the MSU-1.0, MSU-I.1 and MSU-1.2 cells strains which were clonally derived successively by selection for cells which spontaneously exhibited more transformed properties. Each strain is more transformed than the strain from which it is derived, but none is tumorigenic (for details, see Morgan et al., 1991; McCormick and Maher, 1996). The parent of the MSU1 lineage was a normal foreskin-derived fibroblast cell line, LGI. The LG1 cells were transfected with a v-myc gene, and a clone expressing v-rnyc protein was selected and grown to senescence. All the cells died except for a few, probably a single clone, which gave rise to the immortal MSU-1.0 cell strain. These studies indicate that, in addition to myc up-regulation, at least one additional genetic change was required for immortalization. While the parental LG1 cell line displays low level expression of MMP-7, MMP-11 and MMP-13, all of the cell strains derived from LGI express high levels of MMP-7, MMP-11 and MMP-13 (Table IV). Since, as noted above, immortalization of the LG1 derived cells involved both v-myc expression and one or more additional genetic changes, it is not possible to determine whether v-myc alone is responsible for the up-regulation

492

T.A. Giambernardi et al.

of MMP-7, MMP-11 and MMP-13. It is interesting to note that the normal fibroblasts, WI-38 and IMR-90, both express low to negligible levels of MMP-7, MMP11 and MMP-13; however, SV40 transformation and an immortalization event (Shay et al., 1993) again lead to up-regulation of MMP-7 and MMP-13. Note that the MRC-5 fibroblast cell line did not fit the pattern noted with other fibroblastic cell lines. Effect of ras, sis, and fes on M M P expression By Northern analysis, it has been demonstrated that rat embryo cells, which do not express MMP-9, will make MMP-9 mRNA following transformation by Hras or v-myc (Bernhard et al., 1994). MMP-9 expression was found to correlate with invasiveness of tumors (Sato et al., 1992; Sato and Seiki, 1993), and MMP-9 expression may thus indicate that a tumor has progressed toward malignancy. This study supports the observation that increased expression of MMP-9 is found in cells exhibiting high expression of the H-ras oncoprotein. For example, the parental MSU-I.1 cell line expresses low levels of MMP-9; however, the H-ras transformed MSU1.1 derivatives (PH.2M, PH.2MT, PH.3, PH.3T and CL141.JM13) express high levels of MMP-9. The above H-ras transformed cells express eight to 30 times more H-ras oncoprotein than all the H-ras, N-ras and v-K-ras proteins taken together (Hurlin et al., 1989, and J.J. McCormick, unpublished studies). When the same H-ras oncogene is expressed under the control of its normal promoter (resulting in the L98 cell strain), the ras oncoprotein is expressed at only 1.0-1.5 times the level of the total ras protein in the parental MSU-I.1 cell. Note that the L98 cells are not tumorigenic; however, the L98 derivative strains (CCL83.1 and CCL140.14) do not exhibit up-regulation of MMP-9 but are highly tumorigenic in athymic mice (Senoir et al., 1996). It should be noted that the cell strain DY620.10C3/T, which was transformed by transfection of the v-sis oncogene, also exhibits high levels of MMP-9. This is not unexpected, since this cell strain makes high levels of v-sis protein (Yang et al., 1994), and the v-sis gene acts upstream of ras in the same signal transduction pathway. Transfection of the v-fes gene into MSU-I.I or L98 did not result in changes in expression of any MMP gene studied. A v-myc and H-ras transformed cell line, PH3M, expressed a high level of MMP-12 (metalloelastase). Since no other similarly oncogene transformed cell strain expressed MMP-12, this example of MMP-12 expression may have resulted from clonal variation.

Effects of SV40-transformation on M M P expression SV40-transformed and immortalized fibroblast lines exhibit altered expression of MMP-3, -7, -9, -11 and -13 in some, but not all, cases. Up-regulation of MMP-7 and MMP-13 mRNA expression was observed in SV40transformed IMR-90 and WI-38 cells but was not observed in MRC-5. Differences in the expression of MMP mRNAs following SV40 transformation may be due to the fact that SV40 immortalization of cells requires at least one additional event which occurs during the crisis period leading to immortalization (Shay et al., 1993). Alternatively, since IMR-90, WI-38 and MRC-5 were not clonally derived, the transformation event may have led to the immortalization of a clone with a different pattern of MMP expression.

Inappropriate expression of MMPs in tumor cells Wound healing and the involution of the mammary gland are examples of scheduled cellular events which are accompanied by MMP expression (Singh and Foster, 1989; Lund et al., 1996). It is now postulated that a factor in the progression of cells toward malignancy is the developmentally unscheduled or inappropriate expression of one or more MMPs (MacDonald and Steeg, 1993; Hopkin, 1996). As indicated below, this study has revealed additional examples of altered different repertoires of MMP mRNAs. For example, it was found that the relatively non-invasive mammary tumor cell line, MCF-7 (Thompson et al., 1992; Wang et al., 1996), only produced two nearly ubiquitously expressed membrane-type MMPs (MMP-15 and MMP-16); however, we found that the invasive MCF-7 variant, MCF7M/Adr (Pulyaeva et al., 1997), expressed mRNAs for MMP-1, MMP-2, MMP-9, MMP-12. and MMP-14 (Table II). The invasive and non-invasive MCF-7 variants have previously been shown to differ in the expression of several other genes. For example, the MCF7M/Adr variant has been shown to differ from MCF-7 by being estradiol independent for growth, estrogen-receptor negative, tamoxifen resistant, vimentin positive, invasive in both the matrigel and nude mouse assays, and by possessing a transmembrane receptor of the PMP22 family (Schiemann et al., 1997). The MCF7M/Adr variant has also been reported previously to express both MMP-2 and MMP-9 (Pulyaeva et al., 1997h our confirmation of this observation, plus the finding of MMP-I, MMP-12 and MMP-14 expression in the invasive variant, may account for its invasive characteristics (Table II). Several other differences in MMP expression

M M P Overview between invasive and non-invasive cells were also observed in this study. For example, the highly invasive m a m m a r y tumor, M D A - M B - 2 3 1 ( T h o m p s o n et al., 1992; Hansen et al., 1996; Wang et al., 1996), elaborated eight M M P mRNAs. Similarly, the relatively noninvasive prostate cell line L N C a P (Hoosein et al., 1996; Leyton et al., 1996) only produced three M M P s , while the invasive prostate cell line PC-3 (Hoosein et al., 1996; Leyton et al., 1996) made ten MMPs. In conclusion, m a n y studies using variants of subtractive library hybridization and differential display-PCR (DD-PCR) have been conducted which are aimed at the identification of genes expressed in t u m o r cells but not in their normal counterparts (Liang et al., 1992; Sager et al., 1994; Diatchenko et al., 1996). This study, which presents an overview of M M P expression, indicates (a) that m a n y transformed cells display alterations in M M P expression and (b) that the inappropriate expression of M M P s m a y be a major event in the evolution of cells to a malignant phenotype.

Acknowledgements This study was supported in part by grants DE08144, AGl1026 and CA60907 from the National Institutes of Health.

References Abe, T., Mori, T., Kohno, K., Seiki, M., Hayakawa, T., Welgus, H.G., Hori, S. and Kuwano, M. : Expression of 72 kDa type IV collagenase and invasion activity of human glioma cells. Clin. Exp. Metastasis 12: 296-304, 1994. Azzam, H.S., Arand, G., Lippman, M.E. and Thompson, E.W.: Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production. J. Nat. Cancer Inst. 85: 1758-1764, 1993. Basset, P., Wolf, C., Rouyer, N., Bellocq, J., Rio, M. and Chambon, P.: Stromelysin-3 in stromal tissue as a control factor in breast cancer behavior. Cancer 74: 1045-1049, 1994. Belaaouaj, A., Shipley, J.M., Kobayashi, D.K., Zimonjic, D.B., Popescu, N., Silverman, G.A. and Shapiro, S.D.: Human macrophage metalloelastase. Genomic organization, chromosomal location, gene linkage, and tissue-specific expression. J. Biol. Chem. 270: 14568-14575, 1995. Bernhard, E.J., Gruber, S.B. and Muschel, R.J.: Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proc. Natl. Acad. Sci. USA 91: 4293-4297, 1994. Birkedal-Hansen, H.: Proteolytic remodeling of extracellular matrix. Curr. Opin. Cell Biol. 7" 728-735, 1995. Boyd, M.R. and Paull, K.D.: Some practical considerations and applications of the National Cancer Institute in vitro anticancer drug discovery screen. Drug Develop. Res. 34. 91-109, 1995.

493

Buttice, G. and Kurkinen, M.: A polyomavirus enhancer Abinding protein-3 site and Ets-2 protein have a major role in the 12-O-Tetradecanoylphorbol-13-acetate response of the human stromelysin gene. J. Biol. Chem. 268: 7196-7204, 1993. Buttice, G. and Kurkinen, M.: Oncogenes control stromelysin and collagenase gene expression. Contrib. Nephrol. 107: 101-107, 1994. Callaghan, M.M., Lovis, R.M., Rammohan, C., Lu, Y. and Pope, R.M.: Autocrine regulation of collagenase gene expression by TNF-alpha in U937 cells. J. Leukocyte Biol. 59: 125-132, 1996. Cao, J., Sato, H., Takino, T. and Seiki, M.: The C-terminal region of membrane type matrix metalloproteinase is a functional transmembrane domain required for pro-gelatinase A activation. J. Biol. Chem. 270: 801-805, 1996. Chang, J.C., Wysocki, A., Tchou-Wong, K.M., Moskowitz, N., Zhang, Y. and Rom, W.N.: Effect of Mycobacterium tuberculosis and its components on macrophages and the release of matrix metalloproteinases. Thorax 5 I: 306-311, 1996. Chen, D. and Klebe, R.J.: Controls for validation of relative reverse transcription-polymerase chain reaction (RT-PCR) assays. PCR Meth. Applic. 3: 127-129, 1993. Chen, D., Magnuson, V.L., Hill, S., Arnaud, C., Steffensen, B. and Klebe, R.J.: Regulation of integrin gene expression by substrate adherence. J. Biol. Chem. 267: 23502-23506, 1992. Chen, D., Magnuson, V.L., Steffensen, B. and Klebe, R.J.: Use of stock solutions to simplify mRNA quantitation by reverse transcription-polymerase chain reaction (RT-PCR) assays. PCR Meth. Applic. 2: 351-353, 1993. Chomczynski, P. and Sacchi, N.: Single-step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. Diatchenko, L., Lau, Y.-EC., Campbell, A.P., Chenchik, A., Moqadam, E, Huang, B., l_.ukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D. and Siebert, ED.: Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93: 1-6, 1996. Fessler, L.I., Duncan, K.G., Fessler, J.H., Salo, T. and Tryggvason, K.: Characterization of the procollagen IV cleavage products produced by a specific tumor collagenase. J. Biol. Chem. 259: 9783-9789, 1984. Freije, J.M.P., Diez-Itza, I., Balbin, M., Sanchez, L.M., Blasco, R., Tolivia, J. and Lopez-Otin, C.: Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas. J. Biol. Chem. 269: 16766-16733, 1996. Frisch, S.M. and Morisaki, J.H.: Positive and negative transcriptional elements of the human type IV collagenase gene. Mol. Cell. Biol. 10: 6524-6532, 1990. Fry, D.G., Milam, L.D., Dillberger, J.E., Maher, V.M. and McCormick, J.J.: Malignant transformation of an infinite life span human fibroblast cell strain by transfection with v-Kiras. Oncogene 5: 1415-1418, 1990. Gaire, M., Magbanua, Z., McDonnell, S., McNeil, L., Lovett, D.H. and Matrisian, L.M.: Structure and expression of the human gene for the matrix metalloproteinase matrilysin. J. Biol. Chem. 269: 3032-3040, 1994. Graham, C.H., Connelly, I., MacDougall, J.R., Kerbel, R.S., Stetler-Stevenson, W.G, and Lala, P.K.: Resistance of malignant trophoblast cells to both the anti-proliferative and antiinvasive effects of transforming growth factor-beta. Exp. Cell Res. 214: 93-99, 1994.

494

T . A . G i a m b e r n a r d i et al.

Grant, G.M., Cobb, J.K., Castillo, B. and Klebe, R.J.: Regulation of matrix metalloproteinases following cellular transformation. J. Cell. Physiol. 167:177-183, 1996. Gutman, A. and Wasylyk, B.: The collagenase gene promoter contains a TPA and concogene-responsive unit encompassing the PEA3 and AP-1 binding sites. EMBO I. 9: 2241-2246, 1990. Hagmeyer, B.M., Konig, H., Herr, I., Offinga, R., Zantema, A., VanDerEb, A.J., Herrlich, P. and Angel, P.: Adenovirus E1A negatively and positively modulates transcription of AP-1 dependent genes by dimer-specific regulation of the DNA binding and transactivation activities of .]un. EMBO ,l. 12: 3559-3572, 1993. Hansen, C.M., Frandsen, T.L., Brunner, N. and Binderup, L.: l a,25-dihydroxyvitamin D3 inhibits the invasive potential of human breast cancer cells in vitro. Clin. Exp. Metastasis 12: 195-202, 1996. Hasty, K.A., JeffreB J.J., Hibbs, M.S. and Welgus, H.G.: The collagen substrate specificity of human neutrophil collagenase.,l. Biol. Chem. 262: 10048-10052, 1996. Haupt, L.M., Thompson, E.W., Griffiths, 1..R. and Irving, M.G.: IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line. Biochemi. Mol. Biol. Int. 39: 553-561, 1996. Higashino, E, Yoshida, K., Noumi, T., Seiki, M. and Fujinaga, K.: Ets-related protein EIA-F can activate three different matrix metalloproteinase gene promoters. Oncogene 10: 1461-1463, 1995. Hoosein, N.M., I,ogothetis, C.J. and Chung, 1..W.K.: Differential effects of peptide hormones bombesin, vasoactive intestinal polypeptide and somatostatin analog RC-160 on the mvasive capacity of human prostatic carcinoma cells. J. Urol. 149: 1209-1213, 1996. Hopkin, K.: Tumor evolution: Survival of the fittest cells, l. N I H Res. 8: 37-41, 1996. Houde, M., Tremblay, R, Masure, S., Opdenakker, G., Oth, D. and Mandeville, R.: Synergistic and selective stimulation of gelatinase B production in macrophages by lipopolysaccharide, trans-retinoic acid and CGP 41251, a protein kinase C regulator. Biochim. Biophys. Acta l 310:193-200, 1996. Hurlin, P..l., Mabel, V.M. and McCormick, J.J.: Malignant transformation of hunmn fibroblasts caused by expression of a transfected T24 HRAS oncogene. Proc. Natl. Acad. Sci. USA 86: 187-191, 1989. Imai, S. and Takano, T.: Loss of collagenase gene expression in immortalized clones of SV40 T antigen-transformed human diploid fibroblasts. Biochem. Biophys. Res. Commun. 189: 148-153, 1992. Lacraz, S., lsler, P., Vey, E., Welgus, H.G. and Dayer, J.M.: Direct contact between T lymphocytes and monocytes is a major pathway for induction of metalloproteinase expression. J. Biol. Chem. 269: 22027-22033, 1994. Lambert, C.A., l.efebvre, P.Y., Nusgens, B.V. and Lapiere, C.M.: Modulation of expression of endogenous collagenasc and collagen genes by electroporation: possible involvement of Ca ~+ and protein kinase C. Biochem. 1. 290:135-138, 1993. Leyton, J., Manyak, M.I., Mukheriee, A.B., Miele, L., Mantile, G. and Patierno, S.R.: Recombinant human uteroglobin inhibits the in vitro invasiveness of human metastatic prostate tumor cells and the release of arachidonic acid stimulated by fibroblast-conditioned medium. Cancer Res..¢4: 3696-3699, 1996. Liang, P., Averboukh, L., Keyomarsi, K., Sagm, R. and Pardee, A.B.: Differential display and cloning of messenger RNAs

from human breast cancer versus mammary epithelial ceils. Cancer Res. 52: 6966-6968, 1992. Lin, C.-C., Maher, V.M. and McC()rmick, J.J.: Malignant transformation of human fibroblast strain MSU-I.I by v-fes requires an additional genetic change. Int..l. Cancer 63: 140-147, 1995. l~in, C.-C., Wang, D., Maher, V.M. and McCormick, J.].: Malignant transformation of a human fibroblast cell strain by transfection of a v-fes oncogene but not by transfection of a gag-human c-fes construct. Cell Growth Differ. ¢: 1381-1387, 1994. Liu, X.H. and Rose, D.P.: Suppression of type IV collagenase in MDA-MB-435 human breast cancer cells by eicosapentaenoic acid in vitro and in vivo. Cancer Lett. 92: 21-26, 1995. Lohi, J., Lehti, K., Westermarck, J., K/ih/iri, V.M. and KeskiOja, J.: Regulation of membrane-type matrix metalloproteinase-1 expression by growth factors and phorbol 12myristate 13-acetate. Eur. J. Biochem. 239: 239-247, 1996. Lund, I..R., Romer, J., Thomasset, N., Solberg, H., Pyke, C., BisselI, M.J., Keid, D. and Werb, Z.: Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways. Development 122:181-193, 1996. MacDonald, N.J. and Steeg, RS.: Molecular basis of tumor metastasis. Cancer Surveys 16:175-199~ 1993. Mackay, A.R., Ballin, M., Pelina, M.D., Farina, A.R., Nason, A.M., Hartzlm, J.L. and Thorgeirsson, U.P.: Effect of phorbnl ester and cytokines on matrix metalloproteinase and tissue inhibitor of metalloproteinase expression in tumor and norreal cell lines. Invasion Metastasis 12:168-184, 1992. Masure, S., Billiau, A., Van Damme, ,1. and Opdenakkm, G.: Human hepatoma cells produce an 85 kDa gclatinase regulated by phorbol 12-myristate 13-acetate. Biochim. Biophysi. Acta 1054:317-325, 1990. McCormick, J.J., Kohler, S.K. and Maher, V.M.: Transfection of a myc gene as a means of generating infinite life span human fibroblast strains. Meth. Cell Sci. 17:141-148, 199,5. McCormick, J.J. and Maher, V.M.: Analysis of the multistep nature of human carcinogenesis utilizing human fibroblasts. Radiat. Oncol. Invest. 3:387-391, 1996. Milam, S.B., Magnuson, V i . , Steffensen, B., Chen, D. and Klebe, R.J.: IL-lbeta and prostaglandins regulate integrin mRNA expression. ,I. Cell. Physiol. 149:173-183, 1991. Monsky, W.L., Lin, C.Y., Aoyama, A., Kelly, "I\, Akiyama, S.K., Mueller, S.C., Chen, WT. A potential marker protease of invasiveness, seprase, is localized on mvadopodia of human malignant melanoma cells. Cancer Res. 54: 5702-5710, 1994. Morgan, T.1.., Yang, D., Fr B D.G., Hurlin, P.]., Kohler, S.K., Maher, V.M. and McCormick, J.J.: Characteristics of an infinite life span diploid human fibroblast cell strain and a neardiploid strain arising from a clone of cells expressing a transfected v-myc oncogene. Exp.Cell Res. 197:12,5-136, 1991. Murphy, ,1.1:, MurphB G. and Reynolds, ,I.].: The origin of matrix metalloproteinases and their familial relationships. FEBS Lett. 289: 4-7, 1996. Okada, A., Bellocq, J., Rouyer, N., Cbenard, M., Rio, M., Chambon, P. and Basset, E: Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in stromal cells of human colon, breast, and head and neck carcinomas. Proc. Natl. Acad. Sci. USA 92: 2730-2734, 1995. Panagakos, ES. and Kumar, S.: Modulation of proteases and their inhibitors in immortal human osteoblast-like cells by

M M P Overview tumor necrosis factor-alpha in vitro. Inflammation 18: 243-265, 1994. Pulyaeva, H., Bueno, J., Polette, M., Birembaut, P., Sato, H., Seiki, M. and Thompson, E.W.: MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells. Clin. Exp. Metastasis 15:111-120, 1997. Quantin, B., Murphy, G. and Breathnach, R.: Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members. Biochemistry 28: 5327-5334, 1996. Ray, J.M. and Stetler-Stevenson, W.G.: The role of matrix metalloproteases and their inhibitors in tumour invasion, metastasis and angiogenesis. Eur. Respir. J. 7: 2062-2072, 1994. Ries, C., Lottspeich, E, Dittmann, K.H. and Petrides, RE.: HL60 leukemia cells produce an autocatalytically truncated form of matrix metalloproteinase-9 with impaired sensitivity to inhibition by tissue inhibitors of metalloproteinases. Leukemia 10:1520-1526, 1996. Rose, D.R, Connolly, J.M. and Liu, X.H.: Effects of linoleic acid on the growth and metastasis of two human breast cancer cell lines in nude mice and the invasive capacity of these cell lines in vitro. Cancer Res. .54: 6557-6562, 1994. Saarialho-Kere, U.K., Crouch, E.C. and Parks, W.C.: Matrix metalloproteinase matrilysin is constitutively expressed in adult human exocrine epithelium. J. Invest. Dermatol. 105: 190-196, 1995. Sager, R., Sheng, S., Anisowicz, A., Sotiropoulou, G., Zou, Z., Stenman, G., Swisshelm, K., Chen, Z., Hendrix, M.J.C., Pemberton, P., Rafidi, K. and Ryan, K.: RNA genetics of breast cancer: Maspin as paradigm. Cold Spring Harbor Syrup. Quant. Biol. 59: 537-546, 1994. Sat(), H., Kida, Y., Mai, M., Endo, Y., Sasaki, T., Tanaka, J. and Seiki, M.: Expression of genes encoding type IV collagendegrading metalloproteinases and tissue inhibitors of metalloproteinases in various human tumor cells. Oncogene 7: 77-83, 1992. Sat(), H., Kita, M. and Seiki, M.: v-Src activates the expression of 92-kDa type IV collagenase gene through the AP-I site and the GT box homologous to retinoblastoma control elements. J. Biol. Chem. 268: 23460-23468, 1993. Sato, H. and Seiki, M.: Regulatory mechanism of 92 kDa type IV collagenase gene expression which is associated with invasiveness of tumor cells. Oncogene 8: 395~05, 1993. Sat(), H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E. and Seiki, M.: A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370: 61-65, 1994. Schiemann, S., Ruckels, M., Engelholm, I,.H., Schwirzke, M., Brunner, N. and Weidle, U.H.: Differential gene expression in human mammary carcinoma cells: Identification of a new member of a receptro family. Anticancer Res. 17: 13-20, 1997. Schonthal, A., Herrlich, P., Rahmsdorf, H.J. and Ponta, H.: Requirement for los gene expression in the transcriptional activation of collagenase by other oncogenes and phorbol esters. Cell ~4: 325-334, 1988. Senoir, R.M., Griffin, G.L., Fliszar, C.J., Shapiro, S.D., Goldberg, G.I. and Welgus, H.G.: Human 92- and 72-kilodalton type IV collagenases are elastases. ,l. Biol. Chem. 266: 7870-7875, 1996. Shapiro, S.D., Kobayashi, D.K. and Ley, T.J.: Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages. J. Biol. Chem. 268: 23824-23829, 1993.

495

Shay, J.W., van der Haegen, B.A., Ying, Y. and Wright, W.E.: The frequency of immortalization of human fibroblasts and mammary epithelial cells transfected with SV40 large T-antigen. Exp. Cell Res. 209: 45-52, 1993. Shindoh, M., Higashino,; E, Kaya, M., Yasuda, M., Funaoka, K., Hanzawa, M., Hida, K, Kohgo, T., Amemiya, A., Yoshida, K. and Fujinaga, K.: Correlated expression of matrix metalloproteinases and ets family transcription factor E1A-F in invasive oral squamous-cell-carcinoma-derived cell lines. Am. J. Pathol. 148: 693-700, 1996. Singh, G. and Foster, C.S.: Gross factors m treatment of nonhealing corneal ulcers and recurrent erosions. Cornea 8: 45-53, 1989. Stearns, M.E. and Wang, M.: Immunoassays of the metalloproteinase (MMP-2) and tissue inhibitor of metalloproteinase (TIMP 1 and 2) levels in noninvasive and metastatic PC-3 clones: effects of taxol. Oncology Res. 6: 195-201, 1994. Steffensen, B., Magnuson, V.L., Potempa, C i . , Chen, D. and Klebe, R.J.: Alpha5 integrin subunit expression changes during myogenesis. Biochim. Biophys. Aeta 1137: 95-100, 1992. Sundareshan, R, Koster, J.J., Nagle, R.B. and Bowden, G.T.: Coordinated expression of matrilysin during TPA-induced apoptosis of LNCaP cells: two parallel processes affected by TPA. Cancer Lett. 11.3:17-24, 1997. Thompson, E.W., Paik, S., Brunner, N., Sommers, C.L., Zugmaier, G., Clarke, R., Shima, T.B., Torri, J., Donahue, S., Lippman, M.E., Martin, G.R. and Dickson, R.B.: .I. Cell. Physiol. 150: 534-544, 1992. Tienari, J., Pertovaara, I.., Saksela, O., I.ehtonen, E. and Vartio, T.: Increased expression of the matrix metalloproteinase 2 in differentiating Tera 2 human embryonal carcinoma cells. Int. J. Cancer .56: 219-223, 1994. Tsuchiya, Y., End(), Y., Sat(), H., Okada, Y., Mai, M., Sasaki, T. and Seiki, M.: Expression of type-IV collagenases in human tumor cell lines that can form liver colonies in chick embryos. Int. J. Cancer 56:46-51, 1994. VanWart, H.E. and Birkedal-Hansen, H.: The cysteine switch: A principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family. Proc. Natl. Acad. Sci. USA 87: 5578-5582, 1990. Wang, T.N., Qian, X.-H., Granick, M.S., Solomon, M.R, Rothman, V.L., Berger, D.H. and Tuszynski, G.R: Thrombospondin-I (TSP-1) promotes the invasive properties of hyman breast cancer. J. Surg. Res. 6.3: 39-43, 1996. Welgus, H.G., Jeffre.~; J.J. and Eisen, A.Z.: The collagen substrate specificity of human skin fibroblast collagenase..I. Biol. Chem. 256: 9511-9515, 1996. Weston, C.A. and Weeks, B.S.: bFGF stimulates U937 cell adhesion to fibronectin and secretion of gelatinase B. Biochemi. Biophys. Res. Commun. 228:318-323, 1996. Wilhehn, S.M., Collier; I.E., Kronberger, A., Eisen, A.Z., Mariner, B.L., Grant, G.A., Bauer, E.A. and Goldberg, G.I.: Human skin fibroblast stromelysin: Structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells. Proc. Natl. Acad. 5ci. USA 84: 6725-6729, 1996. Wilhelm, S.M., Collier, I.E., Mariner, B.L., Eisen, A.Z., Grant, G.A. and Goldberg, G.I.: SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages Ipublished erratum appears in J. Biol. Chem. 265: 22570, 1990 ]. J. Biol. Chem. 264: 17213-17221, 1989.

496

T.A. Giambernardi et al.

Wilson, D.M., Yang, D., Dillberger, J.E., Dietrich, S.E., Maher, V.M. and McCormick, J.J.: Malignant transformation of human fibroblasts by a transfected N-ras oncogene. Cancer Res. 50: 5587-5593, 1990a. Wilson, D.M., Yang, D., Dillberger, J.E., Dietrich, S.E., Maher, V.M. and McCormick, J.J.: Malignant transformation of human fibroblasts by a transfected N-ras oncogene. Cancer Res. 50: 5587-5593, 1990b. Yang, D., Kohler, S.K., Maher, V.M. and McCormick, J.J.: v-sis oncogene-induced transformation of human fibroblasts into cells capable of forming benign tumors. Carcinogenesis 15: 2167-2175, 1994. Yang, D., Louden, C., Reinhold, D.S., Kohler, S.K., Maher, V,M. and McCormick, J.J.: Malignant transformation of

human fibroblast cell strain MSU-I.1 by (")-7b,8a-dihydroxy-9a, 10a-epoxy-7, 8,9,10-tetrahydrobenzo[a]pyrene. Proc. Natl. Acad. Sci. USA 89: 2237-2241, 1992. Zucker, S., Lysik, R.M., Malik, M., Bauer, B.A., Caamano, J. and Klein-Szanto, A.J.: Secretion of gelatinases and tissue inhibitors of metalloproteinases by human lung cancer cell lines and revertant cell lines: not an invariant correlation with metastasis. Int. J. Cancer 52: 366-371, 1992. Dr. Robert J. Klebe, Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78284, USA. Received September 16, 1997; accepted November 20, 1997