Stephen W. Klemann

Stephen Klemann

Professor
Department of Biology

Visiting Research Scientist, M. D. Anderson Orlando – Cancer Research Institute – Orlando, FL

B.A.,  Hanover College, 1975
M.S.,  Miami University (Oxford, Ohio), 1978
Ph.D., Miami University (Oxford, Ohio), 1982


Office:  Bush Science Center, room 167
Phone: 407-646-2290
E-Mail: Sklemann@rollins.edu


Courses Taught:

  • General Biology
  • Human Reproduction and Development
  • Cellular Biology
  • Molecular Biology
  • Developmental Biology
  • Contemporary Issues in Biology and Medicine
  • Senior Seminar

 

Professional Interests:

My teaching and research interests lie in the converging disciplines of Developmental, Cellular and Molecular Biology. In addition to participating in the General Biology curriculum, I teach Human Reproduction and Development for general education, and Developmental Biology, Cellular Biology, Molecular Biology, and Seminar courses for our programs in Biology and Biochemistry and Molecular Biology.   For many years my research focused on the cloning, characterization, and engineering of genes expressed by peri-implantation stage ovine and bovine conceptuses and the uterine endometrium.  The products of these genes contribute to the communication that occurs between the conceptus and the female early in pregnancy to ensure that pregnancy is established and maintained.  More recently, I have worked in cancer cell biology examining gene expression in human breast, prostate, as well as head and neck squamous cell carcinomas.  I employ DNA microarray methods to obtain genome wide perspectives on gene expression followed by qRT-PCR methods to quantitatively assess the expression of genes of interest identified by microarrays.  This work, which integrates select Rollins undergraduates, is performed at Rollins and in collaboration with scientists at the Cancer Research Institute, M.D. Anderson-Orlando. 

Description of Current Research: 

TITLE:  “AN ANALYSIS OF GENES EXPRESSED BY HEAD AND NECK SQUAMOUS CELL CARCINOMA AND LEKTI RE-EXPRESSING CELL LINES AS THEY RELATE TO METASTASIS”

The ‘Head and Neck’ group of the M. D. Anderson-Orlando Cancer Research Institute investigates  the metastatic behavior of squamous cell carcinomas (SCC) of the oral-pharyngeal area.  A central component of the group’s work focuses on the involvement of a protease inhibitor (LEKTI: lympho-epithelial Kazal-type inhibitor, encoded by SPINK5: serine protease inhibitor Kazal-type 5) whose expression in SCC is lost.  The significance of this has been hypothesized to be in its putative roles in regulating (inhibiting) proteases that otherwise ‘remodel’ the extracellular matrix (ECM) and modify constituents of the cell surfaces.  If allowed to remodel the ECM without control, cells what are otherwise transformed and cancerous can invade surrounding tissues to form tumors and, more significantly, undergo metastasis to spread to distant sites.  If allowed to modify cell surface constituents, including receptors, signaling into the cells may be altered thereby affecting patterns of gene expression.
The metastatic properties of two SCC cell lines (UMSCC-1 and OSC-19) have been studied fairly extensively in vitro and in vivo.  Both the parental cell lines, as well as versions that have been genetically engineered to re-express LEKTI (in hopes of reducing metastasis) have been studied.  Present evidence suggests that re-expression of LEKTI alters in vitro properties such as colony formation, cell adhesion and migration and in vivo properties of invasion and metastasis, thereby reducing the adverse behavior(s) of SCC.  However, little has been done to meticulously examine the molecular biology of either the parental or LEKTI-expressing versions of these cell lines to understand gene expression in a genome-wide manner.  My involvement in the ‘Head and Neck’ group has been to address the following aim:  To characterize in a systematic manner the changes in gene expression that SCC undergo in their transformation from normal to cancerous phenotype and the manner in which LEKTI re-express may modulate gene expression to return cell behavior to ‘normal’.
I have applied three approaches in the characterization of SCC gene expression:

  1. Genome-wide characterization by microarray analysis,
  2. Semi-quantitative analysis by reverse transcription-polymerase chain reaction (RT-PCR), and
  3. Quantitative analysis by ‘real-time’ RT-PCR.
  1. Both UMSCC-1 and OSC-19 have been examined in a genome wide manner using microarrays in an effort to identify genes of interest that have undergone changes in expression compared to normal human epithelial cells.  In addition, the LEKTI re-expressing versions of these SCC have also been examined in relation to the unmodified (and metastatic) parental types to identify genes whose expression is altered by LEKTI re-expression.  As a consequence of this analysis, a set of 96 genes was selected whose expression in Parental and LEKTI re-expressing SCC cell lines were characterized in a more precise manner.  This gene set encodes a variety of proteases and protease inhibitors (consistent with the background presented above), proteins involved in aspects of intercellular communication and cell cycle control (often associated with cells that are neoplastic), and proteins whose relationship with oncogenesis is obscure.
  2. The expression of 96 genes was characterized by reverse transcription-polymerase chain reaction (RT-PCR) in a semi-quantitative manner (semi-quantitative RT-PCR).  This characterization was completed for Parental and two LEKTI re-expressing versions of UMSCC-1 and OSC-19 SCC cultivated in vitro and in vivo, in the form of orthotopic tumors in a mouse model.  In this way, the behavior of the cell lines under study were assessed as they behave in the laboratory as well as in the oral-pharyngeal environment in which SCC tumors normally develop.  Based on this analysis, a decision was made to focus further attention on the OSC-19 Parental and LEKTI re-expressing cell lines and the expression of genes whose products are the proteases that remodel the extracellular matrix and the inhibitors that regulate protease activity.  It was also decided to extend this analysis to include tumors developed in a mouse flank model that would provide tumors that would not be infiltrated with endogenous murine tissue.
  3. The expression of 36 genes encoding protease and protease inhibitors has been characterized by ‘real-time’ RT-PCR to provide quantitative estimates of their expression (quantitative RT-PCR) in OSC-19 cell lines cultivated in vitro as well as in vivo in both orthotopic and flank tumor models.  Analysis of this large data set is ongoing, but preliminary results suggest that 6 of the genes under consideration undergo changes in expression in concert with LEKTI re-expression.  For instance, the expression of MMP-13 (a matrix remodeling metallopeptidase) and TIMP-1 (a tissue inhibitor of metallopeptoidases) exhibit correlations with LEKTI re-expression.  Thus, it appears that a suite of genes whose products regulate the remodeling and stability of the extracellular matrix is likely under coordinated control.  It is the underlying nature of this linkage – the coordinated control of gene expression in oncogenesis and metastasis – that will be important to understand if we are to develop molecular and pharmaceutical strategies to supplement surgery or replace radio- and chemotherapies that can, in and of themselves, be quite damaging.  As such, one of the logical steps in this research program is be to examine the nature of the linkage where correlations between LEKTI re-expression and the expression of genes of interest (GOI) exist.

NOTE: One of the observations made in phase 2 of this project was that the expression of p53 (a tumor suppressor gene whose expression is lost in a diverse set of cancers including breast, ovarian, and colon) is lost in the Head and Neck SCC that we are investigating.  This is not notable in itself as the loss of p53 expression has been noted in other SCC.  However, of great interest is the additional observation that p53 expression appears to be reactivated once we force the re-expression of LEKTI.  This was an unexpected finding.  If this finding survives scrutiny, then not only do we hypothesize that re-expression of LEKTI may coordinate with the expression of various factors involved in the remodeling and stability of the extracellular matrix, but also results in the restoration of a crucial component of the machinery that regulates the cell cycle.  The loss of this tumor suppressor has been implicated in 50% of human cancers and is frequently referred to as the guardian of the genome.  It induces apoptosis (programmed cell death) when cells are beyond ‘redemption’ (too damaged to save) or induces growth arrest until repairs can be made for cells that can be saved!  In the absence of p53, these regulatory mechanisms cannot operate effectively and cells escape normal cell cycle control – they become transformed and cancerous.  Therefore, the nature of the linkage that exists between the expression of LEKTI and p53 is of great interest, and the subject to an Honors Thesis by Amy Iarrobino, one of our senior Biology majors.